AMH-INH-GNIH TRI-EXPRESSION GENE VACCINE OF IMPROVING FECUNDITY OF ANIMALS, PREPARATION METHOD AND APPLICATION
20210085766 ยท 2021-03-25
Assignee
Inventors
- Liguo YANG (Wuhan City, CN)
- Jiaomei TANG (Wuhan City, CN)
- Qunli ZHOU (Wuhan City, CN)
- Aixin LIANG (Wuhan City, CN)
- Aizhen GUO (Wuhan City, CN)
Cpc classification
A61K39/0006
HUMAN NECESSITIES
International classification
Abstract
The invention provides an AMH-INH-GNIH tri-expression gene vaccine capable of improving fecundity of animals and a preparation method of engineering strain thereof. The engineering strain was deposited in China Center for Type Culture Collection on Aug. 15, 2018, with deposit No.: CCTCC NO:M 2018544. When the engineering strain is used for direct immunization of animals or immunization of animals after being mixed with DNA vaccine adjuvant, the fecundity of the animals can be effectively improved. The tri-expression gene is a tri-expression non-resistant DNA plasmid of Mullerian duct resisting hormone, inhibin and gonadotropin release restraining hormone, which can be used for direct immunization of the animals through mucosa immunization to generate antibodies in the manner of being sprayed to noses, orally administered, blended into feeds and the like. Since the gene vaccine does not contain a resistant gene, exogenous antibiotics do not need to be introduced for screening, and antibiotic residues are not generated. Compared with other gene vaccines which need plasmid extraction and purification, high production cost and inconvenient intramuscular injection, and enable the animals to generate stress response, the gene vaccine of the invention is lower in production cost and more convenient to use, and is free from resistance and injection stress response.
Claims
1. An AMH-INH-GNIH tri-expression gene vaccine for improving the fecundity of animals, including tPA-SAMH gene, tPA-SINH gene and tPA-SRFRP gene in sequence.
2. The tri-expression gene vaccine according to claim 1, wherein the gene sequence of tPA-SINH is shown in SEQ ID NO. 1, the gene sequence of tPA-SRFRP is shown in SEQ ID NO. 2 and the gene sequence of the tPA-SAMH is shown in SEQ ID NO.3.
3. The tri-expression gene vaccine according to claim 1, wherein a 2A peptide is ligated between the tPA-SAMH gene and the tPA-SINH gene, a 2A peptide is ligated between the tPA-SINH gene and the tPA-SRFRP gene, the gene sequence of the 2A peptide is shown in SEQ ID NO.4; and the gene sequence of the 2A peptide is shown in SEQ ID NO.5.
4. An engineering strain containing the AMH-INH-GNIH tri-expression gene vaccine according to claim 1.
5. The engineering strain according to claim 4, wherein the engineering strain is deposited at the China Center for Type Culture Collection on Aug. 15, 2018, with a deposit number: CCTCC NO: M 2018544.
6. A method for preparation of AMH-INH-GNIH tri-expression gene vaccine for improving the fecundity of animals, including the following steps: S1. Construction of dual expression plasmids PVAX-tPA-SAMH-2A-tPA-SRFRP-asd and PVAX-tPA-SINH-2A-tPA-SRFRP-asd; S2. Enzyme digestion of the plasmids obtained in step 51 with Hind III and BamH I, respectively, followed by ligation to obtain a tri-expression plasmid PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd.
7. The method according to claim 6, wherein the plasmid PVAX-tPA-SINH-2A-tPA-SRFRP-asd is constructed according to the following method: S1101, Using PVAX-tPA-SINH-asd as a template to amplify the tPA-SINH gene fragment; in which the gene sequence of the tPA-SINH PCR is shown in SEQ ID NO.1, Using PVAX-tPA-SRFRP-asd as a template to amplify the tPA-SRFRP gene fragment; in which the gene sequence of the tPA-SRFRP gene fragment is shown in SEQ ID NO.2. S1102 Enzyme digesting the PCR products of pVAX-asd and tPA-SRFRF with EcoRI and XhoI, followed by ligation to obtain a plasmid PVAX-tPA-SRFRP-asd; S1103. Enzyme digesting the PCR products of PVAX-tPA-SRFRP-asd and tPA-SINH with BamH I and EcoRI, followed by ligation to obtain a plasmid PVAX-tPA-SINH-tPA-SRFRP-asd; S1104. Enzyme digesting the plasmids PVAX-tPA-SINH-tPA-SRFRP-asd and PUC57-2A-2A with EcoRI, followed by ligation to obtain a plasmid PVAX-tPA-SINH-2A-tPA-SRFRP-asd; wherein the gene sequence of the 2A peptide is shown in SEQ ID NO.5.
8. The method according to claim 6, wherein the plasmid PVAX-tPA-SAMH-2A-tPA-SRFRP-asd is constructed according to the following method: S1201. Enzyme digesting the PCR products of PVAX-tPA-SRFRP-asd and tPA-SAMH with Hind III and Kpn I, followed by ligation to obtain a plasmid PVAX-tPA-SAMH-tPA-SRFRP-asd; the gene sequence of the tPA-SAMH PCR product is shown in SEQ ID NO.3; S1202. Enzyme digesting the plasmids PVAX-tPA-SAMH-tPA-SRFRP-asd and PUC57-2A-2A with Kpn I and BamH I, followed by ligation to obtain a plasmid PVAX-tPA-SAMH-2A-tPA-SRFRP-asd; the gene sequence of the 2A peptide is shown in SEQ ID NO.4; S1203. Enzyme digesting the plasmids PVAX-tPA-SAMH-2A-tPA-SRFRP-asd and PVAX-tPA-SINH-2A-tPA-SRFRP-asd with Hind III and BamH I, followed by ligation to obtain a plasmid PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd.
9. A use of the AMH-INH-GNIH tri-expression gene vaccine according to claim 1 in the preparation of medicines for improving the fecundity of animals.
Description
DESCRIPTION OF THE DRAWINGS
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SPECIFIC MODE FOR CARRYING OUT EMBODIMENTS
[0042] Existing research shows that GnIH may directly or indirectly inhibit follicular development and ovulation. However, the inventors have used GnIH gene vaccine to immunize sheep and mice through a large number of experiments, and found that they can stimulate follicle development and ovulation, and increase litter size. INH is one of the important hormones in the hypothalamic-pituitary-gonadal axis regulation system. It inhibits the synthesis and secretion of FSH in the body through feedback, and has a regulatory effect on follicular development. GnIH at the most upstream level of the HPG axis reduces the circulating levels of gonadotropins (LH and FSH) and gonadal steroids by inhibiting the activity of GnRH neurons. AMH can reduce the conversion of primordial follicles to primary follicles, inhibit the basic and stimulative development of primordial follicles (Nilsson et al., 2007). The ovaries of AMH knockout female mice will show exhaustion of primordial follicles (Durlinger et al., 1999). AMH, INH and GnIH have different inhibitory mechanisms for follicular development. The former directly inhibits the development of small follicles, while the latter two inhibit the development of large follicles by inhibiting the secretion of pituitary FSH; it can be seen from the above that if the three are used in combination, they will inhibit follicular development, reduce reproductive ability, but the inventor of the present invention found that among the three hormones that inhibit follicular development, if only one or two of them are immunoneutralized, the inhibitory effect of the remaining two or one hormone will be enhanced. Therefore, neutralizing the three endogenous hormones at the same time can relieve the inhibitory effects of the above three hormones, thereby improving the effect of follicle development. It is believed that there may be a compensatory effect among INH, GnIH and AMH and it is predicted that neutralizing INH, GnIH and AMH at the same time may have a better effect on improving the fecundity of animals. When it is necessary to construct a plasmid that expresses multiple genes, bicistronic or polycistronic expression vectors can be used. In various strategies for constructing multiple expression genes, the internal ribosome entry site (IRES) is widely used. However, due to the large volume of IRES, usually more than 500 nucleotides, and the large difference in expression levels between the genes before and after (the expression amount of downstream genes is about 10%-50% of the upstream), a new strategy is needed to replace IRES. In the present invention, a large number of studies have found that the self-cleaving 2A peptide is used to replace IRES, because it is small (18-22 amino acids) and has a high enzyme digestion efficiency between the upstream and downstream genes of the 2A peptide (almost equimolar expression happens on upstream and downstream). Therefore, in order to enable the efficient expression of multiple expression genes, this study used 2A peptide as a linker, optimized the gene sequence of 2A peptide, and constructed plasmid pVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd. After a large number of experiments, the sequence of each gene used capable of effective expression is liseted as follows: the gene sequence used for determination of tPA-SINH is shown in SEQ ID NO.1, and the gene sequence used for determination of tPA-SRFRP is shown in SEQ ID NO.2 The gene sequence used by tPA-SAMH is shown in SEQ ID NO.3; the gene sequence used by 2A peptide is shown in SEQ ID NO.4; the gene sequence used by 2A peptide is shown in SEQ ID NO.5.
[0043] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the present invention belong to the scope of the present invention.
[0044] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The reagents or raw materials, unless otherwise specified, all come from commercial channels.
Example 1 Construction of an Eukaryotic Tri-Expression Anti-mllerian Hormone, Inhibin, and Gonadotropin Inhibiting Hormone Plasmid PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd
[0045] 1. Construction of a double expression PVAX-tPA-SINH-2A-tPA-SRFRP-asd plasmid;
[0046] 1) PVAX-tPA-SINH-asd as a template (constructed by Huazhong Agricultural University, see Appendix 1 for specific operations) was used to amplify the tPA-SINH gene fragment; wherein the primers used are shown in Table 1, the amplified tPA-SINH gene fragment was shown in SEQ ID NO.1, PVAX-tPA-SRFRP-asd as a template (constructed by Huazhong Agricultural University, see Appendix 2 for specific operations) was used to amplify the tPA-SRFRP gene fragment; the primers used were shown in Table 1, the amplified tPA-SRFRP gene fragment was shown in SEQ ID NO.2;
[0047] 2) Enzyme digestion of pVAX-asd and tPA-SRFRP PCR products was conducted with EcoRI and XhoI, and plasmid PVAX-tPA-SRFRP-asd was obtained after ligation;
[0048] 3) Enzyme digestion of PVAX-tPA-SRFRP-asd and tPA-SINH PCR products was conducted with BamH I and EcoRI, and plasmid PVAX-tPA-SINH-tPA-SRFRP-asd was obtained after ligation;
[0049] 4) Enzyme digestion of the plasmids of PVAX-tPA-SINH-tPA-SRFRP-asd and PUC57-2A-2A (the target fragments were linked to the PUC57 vector, synthesized by Shanghai Sangon Biotech) was conducted with EcoRI, and the plasmid PVAX-tPA-SINH-2A-tPA-SRFRP-asd was obtained after ligation; the gene sequence of 2A in PUC57-2A-2A was shown in SEQ ID NO.5;
TABLE-US-00001 TABLE1 TheprimersequencesoftPA-SINHandtPA-SRFRP Enzyme Digestion Annealing GeneName PrimerSequence(5-3) Site Size Temperature tPA-SINH SEQIDNO.6:F:5-CGCGGATCCGC BamHI 873bp 58C. CACCATGGATGCAATGAAGAGAGGGC-3 SEQIDNO.7:R:-5-CCGGAATTCTT EcoRI GTCTGTGGCAGTCGGCG-3 tPA-SRFRP SEQIDNO.8:F:5-CCGGAATTCGC EcoRI 867bp 58C. CACCATGGATGCAATGAAGAGAGGGC-3 SEQIDNO.9:R:5-CCGCTCGAGTT XhoI AAATGTATACAAACCTCTGGGGC-3
[0050] 2. Construction of PVAX-tPA-SAMH-2A-tPA-SRFRP-asd plasmid;
[0051] PVAX-tPA-SAMH-asd as a template (constructed by Huazhong Agricultural University, see Appendix 3 for specific operations) was used to amplify the tPA-SAMH gene fragment; the primers used were shown in Table 2, and the amplified tPA-SAMH gene fragment was shown in SEQ ID NO.3,
[0052] 5) Enzyme digestion of the PVAX-tPA-SRFRP-asd and tPA-SAMH PCR products was conducted with Hind III and Kpn I, and the plasmid PVAX-tPA-SAMH-tPA-SRFRP-asd was obtained after ligation;
[0053] 6) Enzyme digestion of PVAX-tPA-SAMH-tPA-SRFRP-asd and PUC57-2A-2A plasmids (the target fragment was linked to the PUC57 vector, synthesized by Shanghai Sangon Biotech) was conducted with Kpn I and BamH I, and the plasmid PVAX-tPA-SAMH-2A-tPA-SRFRP-asd was obtained after ligation; the gene sequence of 2A in PUC57-2A-2A was shown in SEQ ID NO.4;
[0054] 7) Enzyme digestion of PVAX-tPA-SAMH-2A-tPA-SRFRP-asd and PVAX-tPA-SINH-2A-tPA-SRFRP-asd plasmids was conducted with Hind III and BamH I, and the plasmid PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRPRP-asd was obtained after ligation;
TABLE-US-00002 TABLE2 TheprimersequencesoftPA-SAMH Enzyme Gene PrimerSequence Digestion Annealing Name (5'-3') site Size Temerature tPA-SAMH SEQIDNO.10:F:5'-CCGAAGCTTG HindIII 843bp 58C. CCACCATGGATGCAATGAAGAGAGGGC-3' SEQIDNO.11:R:-5'-CCGGGTACC KpnI TTGCTGAAAGATGAGTGTCCCG-3'
[0055] 3. Enzyme digestion of PVAX-tPA-SINH-2A-tPA-SRFRP-asd and PVAX-tPA-SAM H-2A-tPA-SRFRP-asd plasmids was conducted with Hind III and BamH I, respectively, and Enzyme digestion of PVAX-tPA-SAMH-2A-TPA-SRFRP-asd plasmid was conducted to obtain the fragment tPA-SAMH-2A (894 bp), (PVAX-tPA-SINH-2A-tPA-SRFRP-asd was 5512 bp, PVAX-tPA-SAMH-2A-tPA-SRFRP-asd was 4581 bp), in which the total volume of the reaction system was 20 l, which contained:
TABLE-US-00003 DNA plasmid 1 g 10 FastDigest Buffer 2 l Hind III 1 l BamH I 1 l ddH.sub.2O to 20 l
[0056] the reaction was conducted in 37 C. water bath for 1h;
[0057] finally, the enzyme digested products were electrophoresed on a 1% agarose gel with Marker III as the molecular weight standard, and the results of electrophoresis were observed and shown in
[0058] 3) Ligation: the gel recovery kit was used to recover the enzyme digested products (see the OMEGA Gel Extraction Kit instruction manual). After the products were recovered, Takara ligase was used to ligate the target fragment and the vector. The total volume of the ligation system was 10 l:
TABLE-US-00004 10 ligation Buffer 1 l T4 DNA ligation 1 l PVAX-tPA-SINH-2A-tPA-SRFRP-asd 2 l SAMH-2A fragment 6 l
[0059] After overnight at 16 C., the ligation product PVAX-tPA-SAMH-2A-tPA-SRFRP-asd can be used directly for transformation or stored at 20 C. for later use.
[0060] 3) Recombinant plasmid PVAX-tPA-SAMH-2A-tPA-SRFRP-asd was transformed into competent bacteria: the ligation product was transformed into competent bacteria 6097 by heat shock method, the method was given below:
[0061] (1) 10 l of recombinant DNA (PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd) was added to 100 l of the competent cells melted in an ice bath, mixed gently, and placed in an ice bath for 30 minutes.
[0062] (2) the tube was heated in a 42 C. water bath for 90 seconds, then quickly transferred to the tube to an ice bath for 2 minutes. Do not shake the centrifuge tube during this process.
[0063] (3) 900 l of sterile LB medium (without antibiotics) was added to each centrifuge tube, mixed well and placed at 37 C. and incubated at 220 r/min for 45 minutes to resuscitate the bacteria.
[0064] (4) the tube was centrifuged at 1000 rpm for 5 min, the supernatant was aspirated and discarded, and an appropriate amount of the supernatant was left.
[0065] (5) the precipitate was gently blowed up, mixed well, and transferred to LB solid medium (without antibiotics), and the cells were evenly spread. the plate was placed in a 37 C. incubator, inverted after 30 minutes, and incubated at 37 C. overnight.
[0066] 4) Screening, identification and sequencing of PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd positive clones: the positive clones were picked from the plates in the previous step (PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd), and inoculated in LB liquid culture medium, and the plasmid was was extracted with plasmid mini kit, enzyme digested with restriction enzymes HindIII/BamHI, HindIII/EcoRI and HindIII/XhoI, and reacted at 37 C. for 1 h, and the results of enzyme digestion was observed by 1.0% agarose gel electrophoresis. The results were shown in
Example 2: Preparation of Eukaryotic Tri-Expression Anti-mllerian Hormone Inhibin Gonadotropin Release-Inhibiting Hormone Plasmid PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd C500 Competent Engineering Strain
[0067] 1. Preparation of Competent Cells
[0068] A sterile inoculating loop was used to pick the cryopreserved Salmonella cholerae suis C500 to streak on the LB agar plate containing DAP (50 g/ml), and streak on the LB agar plate without DAP as a control, and cultured overnight at 37 C. The next day, a single colony that grew well was picked and placed in 5 ml LB liquid medium containing DAP (50 g/ml), and cultured overnight at 37 C. under shaking. 3 ml of the activated culture was inoculated into 300 ml of LB liquid medium containing DAP (50 g/ml), and cultured under shaking at 37 C. for 2.5-3 hours to achieve an OD600 value of about 0.5. The strain culture was poured into a pre-cooled sterile centrifuge bottle under aseptic conditions, placed in an ice bath for 30 minutes and centrifuged at 4 C. at 5000 rpm/min for 10 minutes, and the supernatant was completely discarded, and the strain precipitants were gently suspended with 10 ml of ice-pre-cooled 10% glycerol solution, placed in an ice bath for 10 min, and centrifuged at 4 C. at 5000 rpm/min for 10 min, and the supernatant was discarded. Repeatedly, the strain precipitants were gently suspended with 10 ml of ice-pre-cooled 10% glycerol solution, placed in an ice bath for 10 min, and centrifuged for 10 min, the supernatant was discarded, and finally the strain precipitants were resuspended with 10 ml of ice-pre-cooled 10% glycerol, and the suspended competent cells were divided into 80 l and used immediately for electrotransformation or stored in a 80 C. refrigerator for later use.
[0069] 2. Electrotransformation of C500 with Plasmid
[0070] 20 l of the plasmid was added to fresh 80 l of competent cells, mixed and pre-cooled on ice for 30 min. The electroporator parameters were set as follows: voltage 1.8 KV, time 4 ms-6 ms. The cooled mixture was added to the electric cup, the electric shock was given after the water was wiped off outside of the cup, the SOC medium was immediately added and resuscitates at 37 C. on a shaking table for 45 minutes. After centrifuge at 8000 rpm/min for 1 min, the supernatant was aspirated and discarded, and the rest was spread on a MacConkey solid plate preheated in an incubator, and incubated overnight at 37 C.
[0071] 3. PCR Identification
[0072] A single colony from the MacConkey plate was added in LB medium without any antibiotics, and PCR method was used to identify and screen positive clones, mainly for detection of Crp, Asd, InvA, INH-RFRP, AMH-INH genes, InvA, Crp and Asd were detected whether they were the target strains; AMH-INH and INH-RFRP were detected whether the target strains had been transfected with the target plasmid. The 20 l PCR reaction system used contained upstream and downstream primers 1 l/each (see Table 3 for primer sequences), 2Taq PCR Mix 10 l, template 2 l, and ddH2O 6 l. The reaction program was: pre-denaturating at 94 C. for 4 min, then denaturating at 94 C. for 40 sec, annealing at 58 C. for 30 sec, extending at 72 C. for 50 sec, totalling 35 cycles, and finally extending at 72 C. for 10 min. The results were shown in
TABLE-US-00005 TABLE3 Primercharacteristicsofgenescloned Gene Name PrimerSequence(5-3) Size Crp SEQIDNO.12:F:5-TACG 599bp CGCATACAACAAAAGTCGC-3 SEQIDNO.13:R:-5-GCC ATTCTGACGGAATTAACGGG-3 Asd SEQIDNO.14:F:5-TTGC 315bp TTTCCAACTGCTGAGC-3 SEQIDNO.15:R:-5-TCC TATCTGCGTCGTCCTAC-3 InvA SEQIDNO.16:F:5-CAGG 580bp ATACCTATAGTGCTGC-3 SEQIDNO.17:R:-5-CGC ACCGTCAAAGGAACCGT-3 INH- SEQIDNO.18:F:5-TATG 1027bp RFRP TCCACCGCCCCTCTG-3 SEQIDNO.19:R:5-AATG TATACAAACCTCTGGGGCA-3 AMH- SEQIDNO.20:F:5-ATGA 1010bp INH GGGAAGAGGTCTCCAATA-3 SEQIDNO.21:R:-5-TCT TGTCTGTGGCAGTCGGC-3
[0073] 4. Plasmid extraction by alkaline lysis method
[0074] (1) a single colony from the MacConkey plate was placed in 5 ml of LB medium without any antibiotics, and cultured overnight at 37 C. under shaking at 220 rpm/min.
[0075] (2) 2 ml of strain solution was transferred into a 2 ml of EP tube, centrifuged at 13000 rpm for 1 min, and the supernatant was discarded.
[0076] (3) the precipitate was suspended in 100 l of ice-cold Solution I, vortexed and shaked until the strains were fully suspended.
[0077] (4) 200 l of newly prepared Solution II was added, immediately inverted and mixed well, and placed in ice bath for 5 minutes. 150 l of ice-cold Solution III was added, gently mixed upside down, and then placed in ice bath for 5 minutes.
[0078] (5) the mixture was centrifuged at 13000 rpm for 5 minutes, and the supernatant was transferred to a new EP tube.
[0079] (6) an equal volume of phenol/chloroform/isoamyl alcohol mixture (25:24:1 by volume) were added and mixed thoroughly.
[0080] (7) the mixture was centrifuged at 13000 rpm for 5 minutes, the upper aqueous phase was carefully aspirated, the remaining was transferred to a new EP tube, 2 volumes of absolute ethanol was added, and the mixture was precipitated at 20 C. for 30 minutes.
[0081] (8) the mixture was centrifuged at 13000 rpm for 10 min, the supernatant was discarded, and the precipitate was washed once with 75% ethanol.
[0082] (9) the mixture was centrifuged at 13000 rpm for 5 min, and the supernatant was discarded.
[0083] (10) the EP tube was kept at room temperature for a few minutes, an appropriate amount of TE was added, the precipitate was resuspended, and kept at 56 C. for 30 minutes.
[0084] 5. DNA product purification (refer to Tiangen manual for operation)
[0085] (1) 500 l of balance solution BL was added to the adsorption column CB2, centrifuged at 13,000 rpm for 1 min, the waste liquid in the collection tube was discarded, and the adsorption column CB2 was put back into the collection tube.
[0086] (2) the plasmid to be purified was added, 5 times the volume of binding solution PB was added thereto, and mixed well.
[0087] (3) the solution obtained in the previous step was added to an adsorption column CB2 (the adsorption column was placed in the collection tube), placed at room temperature for 2 minutes, centrifuged at 13,000 rpm for 1 minute, the waste liquid in the collection tube was discarded, and the adsorption column CB2 was put into collection tube. Note: the volume of the adsorption column was 800 l. If the sample volume was larger than 800 l, it can be added in batches.
[0088] (4) 600 l of rinsing solution PW was added to the adsorption column CB2 (please check whether absolute ethanol has been added before use), kept for 2-5 minutes and then centrifuged at 13,000 rpm for 1 minute, the waste in the collection tube Liquid was discarded, the adsorption column CB2 was put into the collection tube.
[0089] (5) step 4 was repeated.
[0090] (6) the adsorption column CB2 was put back into the collection tube, centrifuged at 13,000 rpm for 2 minutes, and the rinse liquid was removed as far as possible. The adsorption column CB2 was placed at room temperature for a few minutes, and dried thoroughly to prevent the residual rinsing solution from affecting the next experiment.
[0091] (7) the adsorption column CB2 was put into a clean centrifuge tube, and 30-50 l elution buffer EB was dropwise added into the middle of the adsorption membrane, and the centrifuge tube was placed at room temperature for 2 minutes, and centrifuged at 13,000 rpm for 2 min to collect the DNA solution.
[0092] 6. Enzyme digestion of DNA product after purification
[0093] Enzyme digestion with restriction endonuclease HindIII/XhoI was conducted at 37 C. for 1 h, followed by 1.0% agarose gel electrophoresis and observation of the enzyme digestion results, the results were shown in
[0094] The results showed that the prepared plasmid contained tri-expression recombinant plasmids (PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRPRP-asd) of anti-mllerian hormone, inhibin and gonadotropin inhibiting hormone, it was deposited in the China Center for Type Culture Collection on Aug. 15, 2018, with a classification name: Salmonella enterica C500 (PVAX-SAMH-2A-SINH-2A-SRFRP-asd), and deposit number: CCTCC NO: M 2018544. PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd plasmid can be obtained by extraction by SDS alkaline lysis method.
Example 3: Detection of In Vitro Expression of Eukaryotic Tri-Expression Anti-mllerian Hormone, Inhibin, Gonadotropin Inhibiting Hormone Plasmid PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd Plasmid
[0095] Detection of transcription level of PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd plasmid transfected cells: a plasmid extraction kit was used to extract the plasmid, when the monolayer of Hela cells (cell line of human cervical cancer cells) grew to 60%-70%, the Hela cells were transfected with Lipofectamine 3000 liposome transfection kit, followed by digestion of the cells with pancreatin and collection of cells after 48 hours. mRNA of the cells was extracted with Trizol, and cDNA was obtained by reverse transcription, which was amplified according to the primers of anti-mllerian hormone, inhibin and gonadotropin release inhibitor (see Table 3).
[0096] The cDNA obtained by reverse transcription was used as a template, the primers of AMH-INH and INH-RFRP (see Table 3) were used to amplify the two target fragments of AMH-INH and INH-RFRP. 1% agarose electrophoresis detection found 1010 bp (AMH-INH) and 1027 bp (INH-RFRP) fragments, shown in
Example 4: Application of Eukaryotic Tri-Expression Non-Resistant DNA Plasmids of Anti-Mllerian Hormone, Inhibin, and Gonadotropin Inhibiting Hormone in Promoting the Fecundity of Animals
[0097] Our laboratory used constructed non-resistant screened vaccines (PVAX-tPA-SAMH-asd, PVAX-tPA-SINH-asd, PVAX-tPA-SRFRP-asd) to immunize mice, proving that it can increase the litter size of mice. Anti-mllerian hormone, inhibin and gonadotropin inhibiting hormone gene vaccine C500 (PVAX-tPA-SAMH-asd, PVAX-tPA-SINH-asd, PVAX-tPA-SRFRP-asd with their respective non-resistance)) was used a a positive control to compare the immune effects of the new tri-gene vaccine PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd that was simultaneously against anti-mllerian hormone, inhibin and gonadotropin inhibiting hormone, to identify the immune effect of the vaccine, and tried to promote the application of a new type of tri-expression anti-mllerian hormone, inhibin and gonadotropin release inhibiting hormone gene vaccine in the production. The specific operations were as follows:
[0098] 1 Materials and methods
[0099] 1.1 Plasmids and Strains
[0100] Plasmids PVAX-tPA-SAMH-asd, PVAX-tPA-SINH-asd, PVAX-tPA-SRFRP-asd, PVAX-tPA-SAMH-2A-tPA-SRFRP-asd, PVAX-tPA-SINH-2A-tPA-SRFRP-asd were constructed and stored by our laboratory; at the same time, Salmonella enterica sv. Choleraesuis C500 (PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd) simultaneously against anti-mllerian hormone, inhibin and gonadotropin release inhibiting hormone after identification was stored at 80 C.
[0101] 1.2 Laboratory Animal Feeding and Management
[0102] SPF-grade 5-week-old female Kunming mice were purchased from Hubei Provincial Center for Disease Control and Prevention. After one week of pre-feeding, they were randomly divided into groups and entered the experimental period. They were raised in a special animal room in the laboratory. The feeding temperature was controlled at about 25 C. with standard feed and regular drinking water. The were raised in cages with 5 animals per cage, sanitized and cleaned once a week, and daily observed for food and water intake and health status.
[0103] 1.3 Test Grouping
[0104] The Kunming female mice pre-fed for 1 week were randomly divided into the following 6 groups as shown in Table 4, 20 per group.
TABLE-US-00006 TABLE 4 Types and doses of immunizing vaccines in each group Immunizing Immunizing dose volume Vaccines (CFU) (l) control PBS 200 PVAX-asd 10.sup.10 200 PVAX-tPA-SAMH-asd 10.sup.10 200 PVAX-tPA-SINH-asd 10.sup.10 200 PVAX-tPA-SRFRP-asd 10.sup.10 200 PVAX-tPA-SAMH-2A-tPA- 10.sup.10 200 SRFRP-asd PVAX-tPA-SINH-2A-tPA- 10.sup.10 200 SRFRP-asd PVAX-tPA-SAMH-2A-tPA- 10.sup.10 200 SINH-2A-tPA-SRFRP-asd
[0105] 1.4 Immunization Methods
[0106] 4 hours before immunization, the water and feed were removed from the mice, and 200 l sodium bicarbonate (7.5%) were given by gavage first. After 30 minutes, the mice in the immunized group were given by gavage 200 l vaccine (see Table 3 for details). Two weeks later, the same method was used to boost the immunization, and the mental and physical conditions of the mice were observed for one week after the immunization.
[0107] 1.5 Weighing and Blood Collection of Mice
[0108] The mice were weighed at the same time period before immunization, 1 and 2 weeks after immunization, and body weight data were recorded. On the day of immunization and the 8th week after immunization, blood was collected from the tail vein. The blood was collected into a 1.5 ml EP tube containing 20 l heparin sodium anticoagulant/tube, centrifuged at 3000 r/min for 10 minutes, and the upper plasma was carefully aspirated and stored at 20 C. for later use.
[0109] 1.6 Statistics of Litter Size and Litter Weight after Mating of Mice
[0110] Two weeks after the boosted immunization with the vaccine, all female mice were divided into cages (2 per cage) and labeled, and healthy male mice were put into the cages until all female mice became pregnant. The litter size, litter weight and weight of newborn mouse.
[0111] 1.7 Detection of AMH/INH/RFRP Antibody
[0112] The indirect ELISA method was used to detect the production of AMH, INH and RFRP antibodies in mice after immunization. The specific steps were as follows:
[0113] (1) Each well of 96-well microplate was coated with 50 ng/100 l of AMH, INH or RFRP antigen, and incubated overnight at 4 C.
[0114] (2) The reaction solution was discarded, the plate was washed with PBST 3 times, 300 l/well, 3 min each time.
[0115] (3) 200 l/well of blocking solution (1% BSA solution) was added and incubated at 37 C. for 1 h.
[0116] (4) The reaction solution was discarded, the plate was washed with PBST 3 times, 300 l/well, 3 min each time.
[0117] (5) 100 l/well of diluted plasma to be tested was added, negative control wells, non-specific adsorption wells (PBST substituted plasma) and zero control were set, and the plated was incubate at 37 C. for 90 minutes.
[0118] (6) The reaction solution was dicarded, the plate was washed with PBST 5 times, 300 l/well, 3 min each time.
[0119] (7) 100 l/well of goat anti-mouse IgG-HRP (Google, 1:3000 dilution) was added, and reacted at 37 C. for 1 h.
[0120] (8) The reaction solution was discarded, and the plated was washed with PBST 5 times, 300 l/well, 3 min each time.
[0121] (9) 150 l/well of TMB substrate color developing solution was added and reacted for 15 min in the dark.
[0122] (10) 2 mol/L H2SO4 stop solution 50 l/well was added to stop the reaction, and the OD value of each well was measured at 450 nm wavelength within 15 min
[0123] 2 Results and Analysis
[0124] 2.1 Immune Response
[0125] 2.1.1 Anti-AMH Antibody Levels after Immunization of Mice with Different Vaccines
[0126] The attenuated Salmonella C500 strain solution into which the plasmid pVAX-asd, pVAX-tPA-SAMH-asd, pVAX-tPA-SAMH-2A-tPA-SRFRP-asd or pVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd was transformed was used to immunize mice at a dose of 10.sup.10 CFU/ml. Eight weeks after the initial immunization, blood was collected and tested for anti-AMH antibodies. It was found that the experimental groups all produced anti-AMH antibodies, and the difference between the experimental groups was not significant (P>0.05, the results were shown in
[0127] 2.1.2 Anti-INH Antibody Levels after Immunization of Mice with Different Vaccines
[0128] The attenuated Salmonella C500 strain solution into which the plasmid pVAX-asd, pVAX-tPA-SINH-asd, pVAX-tPA-SINH-2A-tPA-SRFRP-asd or pVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd was transformed was used to immunize mice at a dose of 10.sup.10 CFU/ml. Blood was collected 8 weeks after primary immunization and tested for anti-INH antibodies. It was found that the experimental groups all produced anti-INH antibodies, and the difference between the experimental groups was not significant (P>0.05, shown in
[0129] 2.1.3 Anti-RFRP Antibody Levels after Immunized Mice with Different Vaccines
[0130] The attenuated Salmonella C500 strain solution into which the plasmid PVAX-asd, PVAX-tPA-SRFRP-asd, PVAX-tPA-SAMH-2A-tPA-SRFRP-asd, PVAX-tPA-SINH-2A-tPA-SRFRP-asd or PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd was transformed was used to immunize mice at a dose of 10.sup.10 CFU/ml, and blood was collected from 8 weeks after the primary immunization and tested for anti-RFRP antibodies. It was found that the experimental group all produced Anti-RFRP antibodies, and the difference between the experimental groups was not significant (P>0.05, shown in
[0131] 2.2 Comparison of Litter Size and Birth Weight of Mice after Immunization with Different Vaccines
[0132] Statistics of the litter size and birth weight of mice immunized with different vaccines showed that the litter size in the PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd group was significantly higher than that of the PBS group, pVAX-asd group, The pVAX-tPA-SAMH-asd group and pVAX-tPA-SRFRP-asd group (Table 5) and was also higher than that of the pVAX-tPA-SINH-asd group, PVAX-tPA-SAMH-2A-tPA-SRFRP-asd group and PVAX-tPA-SINH-2A-tPA-SRFRP-asd group. Statistics of the birth weight of each group showed that genetically immunized mother mice did not affect the birth weight of newborn mice, and the difference between the groups was not significant (P>0.05).
TABLE-US-00007 TABLE 5 Comparison of litter size and litter weight of mice after immunization with different vaccines Birth Groups Litter size weight (g) Control group 13.6 1.72.sup.cC 1.76 0.09 PVAX-asd 13.66 2.30.sup.cC 1.76 0.12 PVAX-tPA-SAMH-asd 14.35 2.43.sup.bcBC 1.78 0.10 PVAX-tPA-SINH-asd .sup.14.94 2.19.sup.abcABC 1.76 0.12 PVAX-tPA-SRFRP-asd 14.26 1.37.sup.bcBC 1.76 0.13 PVAX-tPA-SAMH-2A-tPA- 15.50 3.14.sup.abABC 1.83 0.10 SRFRP-asd PVAX-tPA-SINH-2A-tPA- 15.44 2.13.sup.abABC 1.79 0.18 SRFRP-asd PVAX-tPA-SAMH-2A-tPA- 15.93 1.90.sup.aAB 1.82 0.12 SINH-2A-tPA-SRFRP-asd Note: The lowercase letters marked on the data in the same column are completely different, indicating that the difference is significant (p < 0.05), otherwise, the difference is not significant (p > 0.05), and the uppercase letters marked on the data in the same column are completely different, indicating that the difference is extremely significant (p < 0.01), and all data are expressed as mean + standard deviation.
[0133] The results indicate that the PVAX-tPA-SAMH-2A-tPA-SINH-2A-tPA-SRFRP-asd DNA vaccine constructed by the present invention can effectively improve the fecundity of animals.
APPENDIX 1 CONSTRUCTION OF VECTOR PVAX-TPA-SINH-ASD
[0134] 1.1.1 Amplification of tPA-SINH Fragment
[0135] The pIRES-tPA-SINH-tPA-SRFRP plasmid was used as a template for PCR amplification. In the 20 L PCR reaction system, the template was 1.5 L, the upstream and downstream primers of the tPA-SINH fragment were each 1 L, 2Taq PCR MIX was 10 L, ddH.sub.2O was 6.5 L. The PCR reaction program included pre-denaturating at 94 C. for 4 min, then denaturating at 94 C. for 40s, annealing at 66 C. for 30s, extending at 72 C. for 1 min, totalling 35 cycles, and finally extending at 72 C. for 10 min and 4 C. for 10 min. The upstream primer of tPA-SINH had a KpnI enzyme digestion site, and the downstream primer had an EcoRI enzyme digestion site. The primer sequence was as follows:
TABLE-US-00008 Enzyme Gene PrimerSequence Digestion Annealing Name (5-3) Site Size Temperature PA- F(SEQIDNO.22): KpnI 879bp 66C. SINH 5-CC5GTACCCCGATGG ATGCAATGAAGAGA-3 R(SEQIDNO.23): EcoRI -5-CGGCGGAATTCTTA AATGTATACTCTGTGG C-3
[0136] 1.1.2 Ligation of tPA-SINH Fragment to pMD19T (Simple)
[0137] After the electrophoresis, the agarose gel of the tPA-SINH fragment was cut from the gel under ultraviolet light, and recovered according to the operating procedures of the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 kit. The specific steps were as follows: 1% agarose gel electrophoresis, a single target DNA band was cut from the agarose gel under ultraviolet light and put into a clean 1.5 mL EP tube, 3 times the mass volume (100 mg=100 L) Buffer GM was added, and mixed well to melt the gel block at room temperature, and the EP tube was flicked to make the gel melt completely. Then the melted liquid was transferred to an adsorption column, centrifuged at 12000 rpm for 1 min, the waste liquid in the collection tube was discarded, 500 L Buffer WB was added, followed by centrifuging at 12000 rpm for 30s, washing twice; finally centrifuge at 12000 rpm for 1 min was conducted to remove trace liquid in the adsorption column. The adsorption column was transferred to a clean 1.5 mL EP tube, 30 L Elution Buffer was added, and the tube was placed at room temperature for 2 min, and centrifuged at 12000 rpm for 1 min to elute the DNA. The purity of the recovery was detected by electrophoresis, and the concentration was measured. The collected DNA solution was used in the next experiment or stored at 20 C. for later use.
[0138] The tPA-SINH fragment was ligated to the pMD19T-simple vector. The specific steps were as follows: calculating the ligation volume ratio between the vector and the target fragment according to the recovered DNA concentration, and using SolutionI to ligate the pMD19T and tPA-SINH fragments. The ligation system was as follows:
TABLE-US-00009 Components Amount(L) pMD19T 0.5 tPA-SINH fragment 4.5 SolutionI 5 Total 10
[0139] The system was mixed well, centrifuged for a short time, and ligation was conducted in a 16 C. water bath overnight.
[0140] 1.1.3 Transformation of Strain
[0141] (1) The competent cell DH5a was taken from 80 C. and thawed in ice bath, 10 L of ligation product was added, mixed gently, and placed in ice bath for 30 min.
[0142] (2) The tube was placed at 42 C., followed by heat shock for 90 seconds, and then it was quickly taken out and placed in ice for 2 minutes. Do not shake the centrifuge tube during this process.
[0143] (3) 400 L of LB liquid medium without antibiotics was added, mixed well and placed at 37 C., cultured under shaking at 200 r/min for 1h to resuscitate the strain.
[0144] (4) The tube was centrifuged at 3000 r/min for 5 min, and 4004, of supernatant was discarded.
[0145] (5) The remaining liquid was gently mixed, evenly spread on the LB solid medium containing Amp antibiotic (50 g/mL) with a spreader, and incubated at 37 C. for 12-14h to observe whether or not transformed colonies grow.
[0146] 1.1.4 Screening and Identification of Positive Clones
[0147] A single colony was taken to place in LB liquid medium containing Amp antibiotics (50 g/mL), cultured under shaking at 37 C. at 200 r/min for about 12 hours, Tiangen kit was used for plasmid extraction, pMD19T-tPA-SINH plasmid was subjected to double enzyme digestion identification with KpnI and EcoRI. The enzyme digestion system was as follows:
TABLE-US-00010 Components Amount(L) recombinant plasmid DNA 2 KpnI 2 EcoRI 2 10 buffer 2 ddH.sub.2O 2 Total 10
[0148] The system was mixed well, centrifuged for a short time, and reacted overnight at 37 C. in a water bath.
[0149] 10 L of digested products were subjected to 1% agarose gel electrophoresis detection to screen out the suspected plasmids of pMD19T-tPA-SINH that were sent to Wuhan Tsingke Innovation Biotechnology Co., Ltd. for sequencing, and the strain solution corresponding to the correct plasmid after sequence alignment was amplified, the plasmid was extracted and stored at 20 C. for later use.
[0150] 1.1.5 Enzyme Digestion and Recovery of Vector pVAX-asd and Plasmid pMD19T-tPA-SINH
[0151] With reference to the Thermo manual with a small modification, the specific steps were as follows: Double enzyme digestion of pVAX-asd plasmid and pMD19T-tPA-SINH plasmid with restriction enzymes KpnI and EcoRI was performed to expose the sticky ends at both ends. The enzyme digestion system was as follows:
TABLE-US-00011 Components Amount(L) pVAX-asd or pMD19T-tPA-SINH 5 KpnI 1 EcoRI 1 10 Buffer 2 ddH.sub.2O 11 Total 20
[0152] The system was mixed well, centrifuged for a short time, and reacted overnight at 37 C. in a water bath.
[0153] After enzyme digestion, the target bands were separated by 1% agarose gel electrophoresis, and the tPA-SINH and linear pVAX-asd fragments were recovered using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0. After recovery, recovery purity was detected by electrophoresis and concentration was detected.
[0154] 1.1.6 Ligation of the tPA-SINH Fragment to the Vector pVAX-Asd
[0155] In accordance with the operation instructions of Thermo T4 Ligase, the specific steps were as follows: Calculating the ligation ratio between the vector and the target fragment based on the detected DNA concentration after recovery, and T4 DNA Ligase was used to ligate pVAX-asd and tPA-SINH fragments. The ligation system was as follows:
TABLE-US-00012 Components Amount(L) tPA-SINH fragment 4 pVAX-asd fragment 1 T4 DNA Ligase 1 10 T4 DNA Ligase Buffer 1 H.sub.2O 3 Total 10
[0156] The system was mixed well, centrifuged for a short time, and ligated overnight at 16 C. in a water bath.
[0157] 1.1.7 Preparation of 6097 Competent Cells (Calcium Chloride Method)
[0158] A sterile inoculation loop was used to take the frozen strain 6097 and streak it on the LB plate containing DAP (50 g/mL), and streak it on the LB plate without DAP as a control, and the plates were incubated overnight at 37 C. The next day, a single colony that grew well was picked and cultured under shaking at 37 C. overnight in 5 mL LB liquid medium containing DAP (50 g/mL). 1 mL of the activated culture was inoculated into 100 mL of LB liquid medium containing DAP (50 g/mL), and cultured under shaking at 37 C. for 2.5-3 hours to make the OD600 value reach about 0.5. The strain culture was poured into a pre-cooled sterile centrifuge tube under aseptic conditions, placed in ice bath for 30 minutes, and centrifuged at 5000 r/min at 4 C. for 10 minutes, and the supernatant was discarded completely. Then the strain precipitate was gently suspended with 10 mL ice-precooled 0.1M CaCl.sub.2, placed in ice bath for 30 min, and centrifuged at 4 C. 5000 r/min for 10 min, the supernatant was discarded completely, finally the precipitate was resuspended with ice-precooled 0.1M CaCl.sub.2, 15% sterile glycerin with final concentration was added and mixed well. The resulting solution was divided into 100 L/tube, which was directly used for transformation or stored in 80 C. refrigerator for later use.
[0159] 1.1.8 Transformation of Strain
[0160] (1) The competent cell 6097 was taken out from 80 C. and thawed in an ice bath, 10 L of ligation product was added, mixed gently, and placed in an ice bath for 30 min
[0161] (2) The tube was placed at 42 C., followed by heat shock for 90 seconds, then the tube was quickly taken out and placed on ice for 2 minutes. Do not shake the centrifuge tube during this process.
[0162] (3) 400 L of LB liquid medium without antibiotics was added, mixed well, and cultured under shaking at 37, 200 r/min for 1h to resuscitate the strain.
[0163] (4) The tube was centrifuged at 3000 r/min for 5 min, and 400 L of supernatant was discarded.
[0164] (5) The remaining liquid was gently mixed, and an LB plate without foreign substances was spread with a spreader, and incubated at 37 C. for 18-20 hours to observe whether or not transformed colonies grow.
[0165] 1.1.9 Screening and Identification of Positive Clones
[0166] A single colony was placed in the LB liquid medium without any foreign substances, cultured under shaking at 37 C. 200 r/min for about 12 hours, Tiangen kit was used for plasmid extraction, and pVAX-tPA-SINH-asd plasmid was subjected to a double enzyme digestion identification with KpnI and EcoRI. The enzyme digestion system was shown as follows:
TABLE-US-00013 Components Amount(L) pVAX-tPA-SINH-asd 8 KpnI 1 EcoRI 1 10 buffer 2 ddH.sub.2O 8 Total 20
[0167] The system was mixed well, centrifuged for a short time, and reacted overnight at 37 C. in a water bath.
[0168] 10 L of digestion products was subjected to 1% agarose gel electrophoresis detection to screen out the suspected plasmids of pVAX-tPA-SINH-asd that was sent to Wuhan Tsingke Innovation Biotechnology Co., Ltd. for sequencing, and the train solution corresponding to the correct plasmids after sequence alignment was amplified, and the plasmid was extracted and stored at 20 C. for later use.
APPENDIX 2 CONSTRUCTION OF VECTOR PVAX-TPA-SRFRP-ASD
[0169] 1.2.1 Amplification of tPA-SRFRP Fragment
[0170] The specific PCR system and procedures were as shown in 1.1.2. The upstream primer of tPA-SRFRP had a KpnI enzyme digestion site, and the downstream primer had an EcoRI enzyme digestion site. The primer sequence was as follows:
TABLE-US-00014 Enzyme Gene Primersequence digestion Annealing name (5-3) site Size temerature tPA- F(SEQIDNO.24): KpnI 879bp 66C. SRFRP 5-CGGTACCCCGATGGA TGCAATGAAGAGA-3 R(SEQIDNO.25): EcoRI 5-GAATTCGCGGCCGCT TAAATGTATACAAACC-3
[0171] 1.2.2 Ligation of tPA-SINH Fragment and pMD19T (Simple)
[0172] Refer to 1.1.2. for specific steps.
[0173] 1.2.3 Transformation of Strain
[0174] Refer to 1.1.3 for specific steps.
[0175] 1.2.4 Screening and Identification of Positive Clones
[0176] Refer to 1.1.4 for specific steps.
[0177] 1.2.5 Enzyme Digestion and Recovery of Vector pVAX-Asd and Plasmid pMD19T-tPA-SINH
[0178] Refer to 1.1.5 for specific steps.
[0179] 1.2.6 Target Gene Ligation
[0180] In accordance with the operation instructions of Thermo T4 Ligase. The specific steps were as follows: Calculating the ligation ratio between the vector and the target fragment according to the DNA concentration detected after recovery, and T4 DNA Ligase was used to ligate pVAX-asd and tPA-SRFRP fragments. The ligation system was shown as follows:
TABLE-US-00015 Components Amount(L) tPA-SRFRP fragment 4.5 pVAX-asd fragment 0.5 T4 DNA Ligase 1 10 T4 DNA Ligase Buffer 1 H.sub.2O 3 Total 10
[0181] The system was mixed well, centrifuged for a short time, and ligated overnight at 16 C. in a water bath.
[0182] 1.2.7 Transformation of Strain
[0183] Refer to 1.1.8 for the specific process.
[0184] 1.2.8 Screening and Identification of Positive Clones
[0185] Refer to 1.1.9 for the specific process.
APPENDIX 3 CONSTRUCTION OF VECTOR PVAX-TPA-SAMH-ASD
[0186] 1.3.1 Synthesis of tPA-SAMH Fragment
[0187] The corresponding base sequence of the screened AMH epitope antigen-encoding gene was found out from NCBI, which was inserted into the 5 end of the hepatitis B surface antigen, the tPA signal peptide was inserted at the 3 end of the hepatitis B surface antigen, and two enzyme digestion sites, BamH I and EcoR I, were added in the upstream and downstream to form a tPA-SAMH fragment, which was sent to Sangon Biotech for synthesis.
[0188] 1.3.2 Enzyme Digestion and Recovery of Vector pVAX-Asd and Plasmid pUC-tPA-SAMH
[0189] Refer to 1.1.5 for the specific process.
[0190] 1.3.3 Target Gene Ligation
[0191] Refer to 1.1.2. for specific steps.
[0192] 1.3.4 Strain Transformation
[0193] Refer to 1.1.8 for specific steps.
[0194] 1.3.5 Screening and Identification of Positive Clones
[0195] Refer to 1.1.9 for the specific process.