Nucleic acid vector

Abstract

The present invention provides a nucleic acid vector referred to as pVec constructed through molecular biotechnologies. pVec contains CMV enhancer/promoter, T7 promoter, 5UTR, MCS, 3UTR, poly A (120A)-TTATT, BGH poly (A) signal, kanamycin resistance gene and pUC origin, etc. So pVec can be used as a vector for both DNA vaccines or therapeutic drugs and mRNA vaccines or mRNA therapeutic drugs. The 5UTR, 3UTR and poly A (120A)-TTATT of pVec can be added to the 5 and 3 ends of the in vitro transcribed mRNA respectively and further stabilize the transcribed mRNA. The present invention also provides the constructed pVec-GM-CSF, pVec-hIL-12 and pVAX1-hIL-12, which are used for evaluating the benefits of pVec.

Claims

1. A pVec comprises a nucleic acid sequence that is identical to or at least 86% sequence identical to SEQ ID NO: 28, wherein said nucleic acid sequence comprises nucleotide sequences of a 5UTR, a 3UTR and polyA (120A)-TTATT.

2. The pVec of claim 1, wherein the nucleotide sequence between AgeI and ClaI sites of the 3UTR is identical to or at least 93% sequence identical to SEQ ID NO: 18.

3. The pVec of claim 1, wherein the nucleotide sequence of the 3UTR is identical to or at least 96% sequence identical to SEQ ID NO: 19.

4. The pVec of claim 1, wherein the nucleotide sequence containing the 3UTR and polyA (120)-TTATT is identical to or at least 96% sequence identical to SEQ ID NO: 20.

5. The pVec of claim 1, wherein the pVec has the size of 3,391 base pairs to accommodate an exogenous sequence.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 pVec vector map

(2) The nucleotide length of pVec: 3391 bp

(3) CMV enhancer: bases 36-415

(4) CMV promoter: bases 416-619

(5) T7 promoter: bases 664-680

(6) 5UTR: bases 702-785

(7) Multiple cloning sites: bases 786-878

(8) 3UTR: bases 885-1149

(9) PolyA: bases 1156-1275

(10) TTATT termination sequence: bases 1276-1280

(11) BGH poly (A) signal: bases 1304-1528

(12) Kanamycin resistance gene: bases 1709-2503

(13) pUC origin: bases 2738-3326.

DETAILED DESCRIPTION OF THE INVENTION

(14) The present invention provides a nucleic acid vector referred to as pVec, which is constructed using conventional molecular biotechnologies through the following steps.

(15) Taking conventional pcDNA3.1 as a template, the fragment containing restriction endonuclease AgeI, ClaI, SacII and SpeI sites (SEQ ID NO: 1) is obtained via polymerase chain reaction (PCR) using the forward primer (SEQ ID NO: 2) and the reverse primer (SEQ ID NO: 3), subcloned after ApaI and PmeI sites of the MCS of pcDNA3.1 and transformed into top10 chemically competent E. coli cells or DH5 alpha competent cells, obtaining pcDNA3.1-MCS-ApaI-PmeI-AgeI-ClaI-SacII-SpeI.

(16) To insert poly A (120A) tail-TTATT sequence in the vector, several synthesized oligonucleotides including polyAF1 (SEQ ID NO: 4), polyAF2 (SEQ ID NO: 5), polyAF3 (SEQ ID NO: 6), polyAR1 (SEQ ID NO: 7) and polyAR2 (SEQ ID NO: 8) are phosphorylated with T4 polynucleotide kinase (New England Biolabs, Catalog #: M0201S) at 37 C. for an hour, denatured at 94 C. for 10 minutes, annealed at room temperature for 30 minutes and ligated with T4 DNA ligase at 16 C. overnight, and then subcloned into dephosphorylated SacII and SpeI sites of pcDNA3.1-MCS-ApaI-PmeI-AgeI-ClaI-SacII-SpeI catalyzed by alkaline phosphatase, calf intestinal [(CIP), New England Biolabs, Cat #: M0290S], obtaining pcDNA3.1-MCS-ApaI-PmeI-AgeI-ClaI-SacII-poly A (120A)-TTATT-SpeI. The nucleotide sequence of the inserted poly A (120A)-TTATT sequence is as set forth in SEQ ID NO: 9.

(17) To insert 3UTR (from human -globin) in the vector, the synthesized oligonucleotides including 3UTRClaIF1 (SEQ ID NO: 10), 3UTRClaIF2 (SEQ ID NO: 11), 3UTRSacIIR1 (SEQ ID NO: 12) and 3UTRSacIIR2 (SEQ ID NO: 13) are phosphorylated, denatured, annealed and ligated with T4 DNA ligase, then subcloned into dephosphorylated ClaI and SacII sites of pcDNA3.1-MCS-ApaI-PmeI-AgeI-ClaI-SacII-poly A (120 A)-TTATT-SpeI, obtaining pcDNA3.1-MCS-ApaI-PmeI-AgeI-ClaI-3UTR (-globin)-SacII-poly A (120 A)-TTATT-SpeI.

(18) To insert another 3UTR (from human -globin) in the vector, the synthesized oligonucleotides including 3UTRAgeIF1 (SEQ ID NO: 14), 3UTRAgeIF2 (SEQ ID NO: 15), 3UTRClaIR1 (SEQ ID NO: 16) and 3UTRClaIR2 (SEQ ID NO: 17) are phosphorylated, denatured, annealed and ligated with T4 DNA ligase, and then subcloned into dephosphorylated AgeI and ClaI sites of pcDNA3.1-MCS-ApaI-PmeI-AgeI-ClaI-3UTR (-globin)-SacII-poly A (120 A)-TTATT-SpeI, obtaining pcDNA3.1-MCS-ApaI-PmeI-AgeI-3UTR (-globin)-ClaI-3UTR (-globin)-SacII-poly A (120 A)-TTATT-SpeI. The percentage identity between a query sequence and a subject is obtained using basic local alignment search tool (BLAST). The nucleotide sequence between AgeI and ClaI sites of the -globin 3UTR is identical or at least 93% identical to SEQ ID NO: 18. The nucleotide sequence of 3UTR (2 -globin) is identical or at least 96% identical to SEQ ID NO: 19 and the nucleotide sequence of 3UTR-poly A (120A)-TTATT is identical or at least 96% identical to SEQ ID NO: 20.

(19) To insert 5UTR in the vector, the oligonucleotides including 5UTRF1 (SEQ ID NO: 21), 5UTRF2 (SEQ ID NO: 22), 5UTRR1 (SEQ ID NO: 23) and 5UTRR2 (SEQ ID NO: 24) designed and synthesized by referencing eukaryotic 18s rRNA sequence are phosphorylated, denatured, annealed and ligated with T4 DNA ligase, and then subcloned into dephosphorylated NheI and AflII sites of pcDNA3.1-MCS-ApaI-PmeI-AgeI-3UTR (-globin)-ClaI-3UTR (3-globin)-SacII-poly A (120 A)-TTATT-SpeI, resulting in 5UTR inserted before NheI and obtaining pcDNA3.1-5UTR-MCS-ApaI-PmeI-AgeI-3UTR (-globin)-ClaI-3UTR (-globin)-SacII-poly A (120 A)-TTATT-SpeI, referred to as pcDNA3.1-5UTR-MCS-3UTR-pA. The nucleotide sequence of 5UTR is as set forth in SEQ ID NO: 25.

(20) To delete SpeI site between BamHI and EcoRI sites of the MCS, pcDNA3.1-5UTR-MCS-3UTR-pA is digested with BamHI and EcoRI, blunted and then self-ligated by head to tail connection, obtaining pcDNA3.1-5UTR-MCS (no SpeI, BamHI/EcoRI)-3UTR-pA.

(21) To replace the ampicillin resistance gene of the vector with a kanamycin resistance gene, the fragment containing MluI-MCS-BbsI region of pcDNA3.1-5UTR-MCS (no SpeI, BamHI/EcoRI)-3UTR-pA is obtained by digesting pcDNA3.1-5UTR-MCS (no SpeI, BamHI/EcoRI)-3UTR-pA with MluI and BbsI, and then subcloned between MluI and BbsI sites of pVAX1, obtaining pVec0-5UTR-MCS (no SpeI, BamHI/EcoRI)-3UTR-pA.

(22) To conveniently replace the kanamycin resistance gene of the vector with other non-bacterial antibiotic resistance genes in the future, the fragment containing BbsI-PacI-KanR-PacI-BspHI region is obtained via PCR by taking pVec0-5UTR-MCS (no SpeI, BamHI/EcoRI)-3UTR-pA as a template and using the forward primer (SEQ ID NO: 26) and the reverse primer (SEQ ID NO: 27), subsequently subcloned into BbsI and BspHI (second BspHI) sites of pVec0-5UTR-MCS (no SpeI, BamHI/EcoRI)-3UTR-pA, achieving pVec1-5UTR-MCS (no SpeI, BamHI/EcoRI)-3UTR-pA (with BbsI-PacI-KanR-PacI-BspHI), referred to as pVec. pVec is deposited as PTA-122648 at the American Type Culture Collection (ATCC).

(23) The complete nucleotide sequence of pVec has been sequenced by US Genewiz Company and has identity or at least 86% identity to SEQ ID NO: 28.

(24) The present invention provides a nucleic acid vector referred to as pVec, which contains CMV enhancer/promoter, T7 promoter, 5UTR, MCS, 3UTR, poly A (120A)-TTATT, bovine growth hormone (BGH) poly (A) signal, kanamycin resistance gene and pUC origin, etc. pVec having the size of 3,391 bp is relatively small so that pVec can accommodate large exogenous gene sequences. The 5UTR sequence of pVec can be added to the 5end of the in vitro transcribed mRNA. The 3UTR and poly A (120A)-TTATT sequence of pVec can be added to the 3end of the in vitro transcribed mRNA. The 5UTR, 3UTR and poly A (120A)-TTATT sequence of pVec can stabilize the in vitro transcribed mRNA, further for making mRNA vaccines or therapeutic drugs. Restriction endonuclease SpeI site of the MCS of pVec is deleted and another SpeI site is inserted after poly A (120A)-TTATT sequence of pVec. Therefore, it is easy to generate the linearized plasmid DNA through SpeI digestion and further produce the in vitro transcribed mRNA. pVec contains CMV enhancer/promoter, MCS, BGH poly (A) signal and kanamycin resistance gene and pUC origin so that pVec can be used as a vector for DNA vaccines or therapeutic drugs in human clinical applications. The 5UTR, 3UTR and poly A (120A)-TTATT sequence of pVec can be added to the 5 and 3 end of the transcribed mRNA in cells so that pVec can stabilize the transcribed mRNA better than other conventional vectors such as pcDNA3.1 and pVAX1. In addition, the kanamycin resistance gene of pVec is flanked by two restriction endonuclease PacI sites and easily replaced with other non-antibiotic resistance genes, further generating the DNA vaccine with the non-antibiotic selection gene.

Example 1: Construction and Expression of pVec-GM-CSF

(25) Taking pCMV-SPORT6-GM-CSF [purchased from Open Biosystems, human granulocyte macrophage colony-stimulating factor (GM-CSF), GenBank accession number: BC108724] as a template, the product obtained by PCR amplification using the forward primer designed and synthesized according to Kozak sequence (the nucleotide sequence is identical or at least 81% identical to SEQ ID NO: 29) and the reverse primer (the nucleotide sequence is identical or at least 93% identical to SEQ ID NO: 30) is subcloned into HindIII and XhoI sites of pVec, which is transformed into E. coli cells (e.g., top10 chemically competent E. coli cells or DH5 alpha competent cells), obtaining pVec-GM-CSF.

(26) pVec-GM-CSF is amplified, purified with Qiaprep spin miniprep kit (Qiagen, Cat #: 27106), and digested with restriction endonuclease SpeI, obtaining the linearized plasmid DNA. A small amount of the above SpeI cut plasmid DNA is used for detecting whether pVec-GM-CSF is completely linearized by 1% agarose gel electrophoresis. The mixture of 100 l SpeI cut plasmid DNA reaction solution with about 500 l Buffer PB is transferred into a spin column, centrifuging for 30 seconds and discarding the effluent (flow-through). Then 750 l Buffer PE is added to the above spin column, centrifuging for 30 seconds and draining the effluent, centrifuging for 1 minute again. The spin column is put into a clean micro-centrifuge tube, adding 30 l H.sub.2O to the spin column, standing for 1 minute and centrifuging for 1 minute. The concentration of the purified linearized pVec-GM-CSF is checked and adjusted to 0.5 to 1 g/l.

(27) The in vitro transcribed GM-CSF mRNA is generated by taking the above purified linearized pVec-GM-CSF as a template and using HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, Cat #: E2040S) and 3-0-Me-m.sup.7G(5)ppp(5)G RNA Cap Structure Analog (ARCA, New England Biolabs, Cat #: S1411S) through the following steps.

(28) In detail, the following reagents are added to a 1.5 ml micro-centrifuge tube at room temperature.

(29) TABLE-US-00001 Nuclease-free water x l 10 X reaction buffer 2 l ATP (100 mM) 2 l 10 mM final UTP (100 mM) 2 l 10 mM final CTP (100 mM) 2 l 10 mM final GTP (20 mM) 2 l 2 mM final ARCA (40 mM) 4 l 8 mM final Template DNA (linearized) x l 1 g T7 RNA polymerase mix 2 l Total reaction volume 20 l

(30) After mixing well and pulse-spinning, the above reaction tube is incubated at 37 C. for 2 hours. To remove the template DNA, 70 l nuclease-free H.sub.2O, 10 l of 10 DNase I buffer and 2 l DNase I (New England Biolabs, Cat #: M0303S) are added to the above reaction tube, incubating at 37 C. for 15 minutes.

(31) Using RNeasy mini kit (Qiagen, Cat #: 74104), the in vitro transcribed GM-CSF mRNA is purified by the following steps.

(32) About 20 to 30 l of the above in vitro transcribed mRNA diluted with nuclease-free H.sub.2O is taken and transferred into a micro-centrifuge tube (nuclease-free). 350 l Buffer RLT containing 1% -mercaptoethanol (-ME) is added to the above tube. After thoroughly mixing with pipette, adding an equal volume of 70% ethanol and mixing again, the above mixture is transferred into a spin column for centrifuging and draining the effluent (flow-through). 700 l Buffer RW1 is added to the above spin column, draining the effluent after centrifugation. 500 l Buffer RPE is added to the above spin column, centrifuging, draining the effluent and repeating twice. After centrifuging for 1 minute, the spin column is transferred into a clean micro-centrifuge tube (nuclease-free) and 30 l nuclease-free H.sub.2O is added to the spin column, standing for 1 minute and then centrifuging. The resulting product is the purified in vitro transcribed GM-CSF mRNA. The concentration of the above mRNA is checked using a nanodrop spectrophotometer and then its quality is detected by 1% formaldehyde agarose gel electrophoresis.

(33) pVec-GM-CSF DNA (5 g) and the in vitro transcribed GM-CSF mRNA (5 g) are respectively electroporated into 110.sup.6 cells (e.g., mouse B16F10 cells or D5LacZ cells, etc.) in a 0.2 cm cuvette at the condition of 350 V and 500 s. The above cells electroporated with the DNA or mRNA are cultured in a cell culture medium at 5% CO.sub.2, 37 C. for 36 hours and the supernatants are respectively collected.

(34) Using human GM-CSF enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, Cat #: 88-8337-22), human GM-CSF expressed in the supernatant is detected by the following steps.

(35) The ELISA plate is coated with 100 l capture antibody diluted with 1 coating buffer at the ratio of 1:250 for each well, sealed and incubated at 4 C. overnight.

(36) After discarding the coating solution, rinsing with wash buffer [1 phosphate-buffered saline (PBS) containing 0.05% Tween-20] 3 times, at least 1 minute each time, and patting dry, 200 l of 1 ELISA/ELISPOT Diluent is added to each well of the above plate, incubating at room temperature for 1 hour.

(37) After washing the plate according to the previous method, 100 l of 1 ELISA/ELISPOT Diluent diluted standard human GM-CSF or 100 l of the collected supernatant is added to each well of the above plate, then sealing and incubating at room temperature for 2 hours.

(38) After washing the plate according to the previous method 3 to 5 times, 100 l of 1 ELISA/ELISPOT Diluent diluted detection antibody is added to each well, then sealing and incubating at room temperature for 1 hour.

(39) After washing the plate according to the above method 3 to 5 times, 100 l of 1 ELISA/ELISPOT Diluent diluted Avidin-horseradish peroxidase (HRP) is added to each well, sealing and incubating at room temperature for 30 minutes.

(40) After washing the plate according to the above method 5 to 7 times, 100 l of 1 tetramethylbenzidine (TMB) solution is added to each well, then incubating at room temperature for 15 minutes.

(41) Then 50 l of 2 M H.sub.2SO.sub.4 stop solution is added to each well of the above plate. The concentration of human GM-CSF expressed in the cell supernatant is determined by measuring optical density (OD) value at 450 nm using a micro-plate reader.

(42) The results show that both the cells electroporated with pVec-GM-CSF DNA and the cells with the in vitro transcribed GM-CSF mRNA can express human GM-CSF. In addition, the cells electroporated with the in vitro transcribed GM-CSF mRNA stored at room temperature for over three weeks can still express GM-CSF.

Example 2: Construction of pVec-hTL-12 and Comparing pVec-hIL-12 with pVAX1-hIL-12

(43) Human interleukin-12 (hIL-12) gene is obtained by digesting pORF-hIL-12 G2 (InvivoGen) with SalI and NheI, and subcloned into XhoI and XbaI sites of pVec, obtaining pVec-hIL-12. Also, hIL-12 gene digested with SalI and NheI is subcloned into XhoI and XbaI sites of pVAX1 (Invitrogen), obtaining pVAX1-hTL-12.

(44) Using the above mentioned method, pVec-hIL-12 and pVAX1-hIL-12 are respectively amplified, purified with Qiaprep spin miniprep kit (Qiagen, Cat #: 27106) and linearized by SpeI digestion, obtaining the corresponding linearized plasmid DNAs. The concentration of the resultant linearized pVec-hIL-12 and pVAX1-hIL-12 is checked and adjusted to 0.5 to 1 g/l.

(45) The in vitro transcribed mRNAs respectively from pVec-hIL-12 and pVAX1-hIL-12 are generated by the previous indicated method. The obtained mRNAs are respectively purified using RNeasy mini kit (Qiagen, Cat #: 74104). The concentration of the mRNAs is checked using a nanodrop spectrophotometer and their quality is detected by 1% formaldehyde agarose gel electrophoresis.

(46) pVec-hIL-12 DNA, the in vitro transcribed hIL-12 mRNA from pVec-hIL-12, pVAX1-hIL-12 DNA and the in vitro transcribed hIL-12 mRNA from pVAX1-hIL-12 (5 g/each) are respectively electroporated into 110.sup.6 cells (such as mouse B16F10 cells or D5LacZ cells, etc.) in a 0.2 cm cuvette at the condition of 350 V and 500 is. The above electroporated cells are cultured in a cell growth medium at 5% CO.sub.2, 37 C. for 36 hours, the supernatants of the above cells are respectively collected.

(47) The collected supernatants are respectively used for detecting human IL-12 expression using human IL-12 ELISA kit (eBioscience, Cat #: 88-7126-88) by the previous mentioned protocol.

(48) The ELISA plate is coated with 100 l capture antibody diluted with 1 coating buffer at the ratio of 1:250 for each well, sealed and incubated at 4 C. overnight.

(49) After discarding the coating solution containing capture antibody, rinsing with wash buffer (1PBS containing 0.05% Tween-20) 3 times, at least 1 minute each time, and patting dry, 200 l of 1 ELISA/ELISPOT Diluent is added to each well of the above plate, then incubating at room temperature for 1 hour.

(50) According to the previous mentioned method, the above plate is washed. 100 l of 1 ELISA/ELISPOT Diluent diluted standard human IL-12 or 100 l of the collected supernatant is added to each well, then sealing and incubating at room temperature for 2 hours.

(51) The plate is washed according to the previous method 3 to 5 times and 100 l of 1 ELISA/ELISPOT Diluent diluted detection antibody is added to each well, then sealing and incubating at room temperature for 1 hour.

(52) The plate is washed according to the above method 3 to 5 times. 100 l of 1 ELISA/ELISPOT Diluent diluted Avidin-HRP is added to each well, then sealing and incubating at room temperature for 30 minutes.

(53) The plate is washed according to the above method 5 to 7 times. 100 l of 1TMB solution is added to each well, incubating at room temperature for 15 minutes.

(54) Then 50 l of 2 M H.sub.2SO.sub.4 stop solution is added to each well of the above plate. Further, the concentration of human IL-12 expressed in the cell supernatant is determined by measuring OD value at 450 nm using a micro-plate reader.

(55) The experiments show that the cells electroporated with pVec-hIL-12 DNA, the in vitro transcribed mRNA from pVec-hIL-12, pVAX1-hIL-12 DNA and the in vitro transcribed mRNA from pVAX1-hIL-12 can express hTL-12 respectively. In addition, pVec-hIL-12 as a template is used for generating the in vitro transcribed mRNA, which has good stability. The amount of hIL-12 expressed by the in vitro transcribed mRNA from pVec-hIL-12 is also higher than that of the in vitro transcribed mRNA from pVAX1-hIL-12.