Recombinant Hansenula polymorpha-based high dosage hepatitis B vaccine

Abstract

Provided is a recombinant Hansenula polymorpha-based high dosage hepatitis B vaccine, an HBsAg pure stock solution yield of a recombinant Hansenula polymorpha fermentation broth used for producing the hepatitis B vaccine being 300 mg/L-400 mg/L.

Claims

1. A hepatitis B vaccine comprising a recombinant Hansenula polymorpha, comprising and expressing hepatitis B surface antigen (HBsAg) of SEQ ID NO: 1, wherein a host Hansenula polymorpha cell line of the recombinant Hansenula polymorpha is HU-11, and the accession number of the host Hansenula polymorpha cell line is CGMCC No.1218, and a disrupted DNA sequence of an orotidine-5-phosphate decarboxylase gene of the host Hansenula polymorpha is SEQ ID NO: 3, wherein the dose of the HBsAg in the hepatitis B vaccine is 40 pg/dose for adults and 20 pg/dose for children, and wherein the pure stock solution yield of the hepatitis B vaccine is 300 mg/L fermentation liquid to 400 mg/L fermentation liquid.

2. The hepatitis B vaccine comprising a recombinant Hansenula polymorpha according to claim 1, wherein the amino acid sequence of the HBsAg expressed by the recombinant Hansenula polymorpha is shown in SEQ ID NO: 2.

3. The hepatitis B vaccine comprising a recombinant Hansenula polymorpha according to claim 1, wherein the HBsAg expressed by the recombinant Hansenula polymorpha is a virus-like particle structure, which is formed by inserting HBsAgs into Hansenula polymorpha lipid, and wherein 9 to 12 among the 14 cysteic acids of the HBsAg form disulfide bonds.

4. The hepatitis B vaccine comprising a recombinant Hansenula polymorpha according to claim 1, wherein the hepatitis B vaccine further comprises an adjuvant; the adjuvant is aluminum adjuvant prepared by in-situ coprecipitation or the direct adsorption; the aluminum adjuvant is preferably prepared by in-situ coprecipitation.

5. The hepatitis B vaccine comprising a recombinant Hansenula polymorpha according to claim 1, wherein the immunization procedure of the high dosage of hepatitis B vaccine is one injection respectively in 0, 1, 2 to 12 months for three needles; the preferably immunization procedure is one injection respectively in 0, 1, and 6 months for three needles.

6. The hepatitis B vaccine comprising a recombinant Hansenula polymorpha according to claim 1, wherein the dosage form of the hepatitis B vaccine is selected form prefilled injection solution, injection solution or lyophilized powder injection; the dosage form is preferably the prefilled injection solution.

7. The hepatitis B vaccine comprising a recombinant Hansenula polymorpha according to claim 1, wherein the hepatitis B surface antigen is adw subtype.

8. A recombinant Hansenula polymorpha, wherein the recombinant Hansenula polymorpha comprises the nucleotide sequence of SEQ ID NO: 1, and the nucleotide sequence of SEQ ID NO: 1 is integrated into the genome of the recombinant Hansenula polymorpha, wherein a host Hansenula polymorpha cell line of the recombinant Hansenula polymorpha is HU-11, and the accession number of the host Hansenula polymorpha cell line is CGMCC No.1218, and a disrupted DNA sequence of an orotidine-5-phosphate decarboxylase gene of the host Hansenula polymorpha is SEQ ID NO: 3.

9. The hepatitis B vaccine comprising a recombinant Hansenula polymorpha according to claim 3, wherein the HBsAg expressed by the recombinant Hansenula polymorpha is a virus-like particle structure, which is formed by inserting HBsAgs into Hansenula polymorpha lipid, and wherein 9 to 12 among the 14 cysteic acids of the HBsAg form disulfide bonds.

10. The hepatitis B vaccine comprising a recombinant Hansenula polymorpha according to claim 3, wherein the hepatitis B vaccine further comprises an adjuvant; the adjuvant is aluminum adjuvant prepared by in-situ coprecipitation or the direct adsorption; the aluminum adjuvant is preferably prepared by in-situ coprecipitation.

11. The hepatitis B vaccine comprising a recombinant Hansenula polymorpha according to claim 4, wherein the hepatitis B vaccine further comprises an adjuvant; the adjuvant is aluminum adjuvant prepared by in-situ coprecipitation or the direct adsorption; the aluminum adjuvant is preferably prepared by in-situ coprecipitation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present disclosure will become more fully understood from the detailed description and the accompanying drawings, in which:

(2) FIG. 1 is a schematic view showing the construction process of plasmid pMPT-HBs adw2.

(3) FIG. 2 is a physical map of plasmid pMPT-HBs adw2;

(4) FIG. 3 is an electrophoresis photograph of a PCR amplification product of the engineered strain obtained by screening from over 100 copies transformant;

(5) FIG. 4 is an electron micrograph of the pure stock solution of recombinant Hansenula polymorpha recombinant HBsAg;

(6) FIG. 5 is a flow chart showing the steps of transformation and screening of recombinant Hansenula polymorpha in Example 2.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

(7) The present disclosure is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Like numbers in the drawings indicate like components throughout the views. As used in the description herein and throughout the claims that follow, unless the context clearly dictates otherwise, the meaning of a, an, and the includes plural reference, and the meaning of in includes in and on. Titles or subtitles can be used herein for the convenience of a reader, which shall have no influence on the scope of the present disclosure.

(8) The terms used herein generally have their ordinary meanings in the art. In the case of conflict, the present document, including any definitions given herein, will prevail. The same thing can be expressed in more than one way. Alternative language and synonyms can be used for any term(s) discussed herein, and no special significance is to be placed upon whether a term is elaborated or discussed herein. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms is illustrative only, and in no way limits the scope and meaning of the present disclosure or of any exemplified term. Likewise, the present disclosure is not limited to various embodiments given herein. Numbering terms such as first, second or third can be used to describe various components, signals or the like, which are for distinguishing one component/signal from another one only, and are not intended to, nor should be construed to impose any substantive limitations on the components, signals or the like.

(9) The Construction of the Hansenula polymorpha Intracellular Plasmid pMPT-02 is the Applicant's Non-Exclusive Proprietary Technology:

(10) 1.5 kb Hansenula polymorpha MOX (methanol oxidase) promoter, 350 bp Hansenula polymorpha MOX (methanol oxidase) terminator, 1.0 kb Hansenula polymorpha autonomous replication sequence HARS, and 1.1 kb Saccharomyces cerevisiae uracil gene ScURA3 were tightly ligated by gene synthesis technology element, and then inserted into the pBluescripII plasmid to construct a shuttle plasmid pMPT-02.

(11) Development of the Host Cell Using the Uracil Auxotrophic URA3-Host Cell Line HU-11:

(12) A recombinant H. polymorpha strain HU-11 (The accession number for the deposit: CGMCC No. 1218. The date of the deposit: Sep. 13, 2004. The name and address of the depository: China General Microbiological Culture Collection Center (CGMCC), No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China) in which the orotidine-5-phosphate decarboxylase gene (HURA3) was disrupted by homologous sequence-mediated homologous integration. Compared with the conventional auxotrophic host strains produced by mutagenesis, the recombinant H. polymorpha strain HU-11 has the characteristics of high genetic stability and low back mutation rate. It was convenient for genetic transformation and screening of recombinant strains, and maintains the wild-type strain. The physiological and biochemical characteristics were beneficial to the culture of recombinant strains and the high expression of foreign proteins, and have high industrial application value. The DNA sequencing result of the disrupted URA3 gene of the Hansenula polymorpha host strain HU11 showed that the five bases of GAAGT were inserted into the 31st base. The insertion of five bases of GAAGT produces a frameshift mutation. The frameshift mutation resulted in a mutation in all of the 254 amino acid codes after the 11th position, and the mutation produced a total of 15 termination codes, indicating that the structural gene of URA3 is no longer re-expressible. The probability that the five bases GAAGT simultaneously produce a back reversion mutation was extremely small. The experimental test also proved that the back mutation rate of the host strain HU11 is zero. This low back-reversion mutation rate of the host strain was particularly advantageous for transformation screening. URA3-ogal deficiency host cell line HU-11 (CGMCC No. 1218) established by gene knock out technology was disclosed in the applicant's previously invention CN1651570A. The DNA sequence in which the disrupted decarboxylase gene (HURA3) was shown in SEQ ID NO:3.

(13) The DNA sequence of HBsAg expression of the recombinant Hansenula polymorpha of the present disclosure was based on the HBsAg adw2 subtypes as shown in SEQ ID NO: 1. The amino acid sequence of the HBsAg is shown in SEQ ID NO: 2.

(14) Construction of the Hansenula polymorpha Intracellular Plasmids pMPT-HBs adw2 (See FIG. 1):

(15) A synthetic nucleotide sequence according to the sequence shown in SEQ ID NO: 1 (hereinafter referred to as HBsAg adw2 gene) was constructed into a glycerol strain containing the HBsAg adw2 gene plasmid; the plasmid after correct sequencing was digested with EcoRI/BamHI, and then 701 bp DNA fragment was obtained.

(16) The correct plasmid pMPT-02 was digested with EcoRI/BamHI, and the vector DNA obtained after the gelatinization was ligated to obtain the Hansenula polymorpha intracellular plasmid pMPT-HBs adw2, and the plasmid pMPT-HBs adw2 was heat shock transformed into E. coli Competent Cell JM109 (Code No. D9052), and then was cultured overnight by plating. Single colonies were selected from the transformation plates, plasmid DNA was extracted and digested with EcoRI/BamHI, and the results of restriction enzyme digestion showed positive clones. Sequencing confirmed that the plasmid pMPT-HBs adw2 was correct.

(17) The HBsAg adw2 gene was inserted into the multiple cloning site of the Hansenula polymorpha expression system intracellular plasmid pMPT-02: between EcoRI and BamHI. The full length of the plasmid pMPT-HBs adw2 is 7665 bp. A schematic diagram of the construction process of plasmid pMPT-HBs-adw2 is shown in FIG. 1. The physical map of the pMPT-HBs adw2 plasmid is shown in FIG. 2.

(18) Construction of Recombinant Hansenula polymorpha Hepatitis B Virus Surface Antigen (HBsAg) Adw2 Subtype Engineering Strain:

(19) In order to construct the recombinant Hansenula polymorpha hepatitis B virus surface antigen (HBsAg) adw2 subtype engineering strain, the cell electroporation technology developed by the applicant was applied. The RC pulse: amplitude 1500V, capacitance 22 F, and time constant 3-5 ms electric shock 1 time, adopted the pMPT-HBsadw2 plasmid transformed into Hansenula polymorpha cells of the HU-11 strain (CGMCC No. 1218) from which the URA3-gene was knocked out. The single colony transformants were picked up on the MD selection culture plate and transferred to the MD liquid medium for continuous subculture. The adw2 subtype HBsAg gene and the corresponding regulatory components were multi-copy and heterologously integrated into the host Hansenula polymorpha cell chromosome. After a single colony of more than one thousand transformant single colonies, the following three steps were screened:

(20) (1) Clonal strains with large single colonies and fast cell growth have a high probability of multiple copies.

(21) (2) The PCR technique was used to compare the electrophoretic band luminance of the HBsAg gene and the single copy number MOX (methanol oxidase) gene, and the HBsAg gene copy number was determined semi-quantitatively.

(22) (3) The expression level of HBsAg released after methanol-induced and shake flask culture for 72 hours was detected.

(23) The application of PCR technology to transformants screening was a new creation of this application. The multiple copies of the foreign gene HBsAg are determined and heterologously integrated in the Hansenula polymorpha chromosome, while the MOX gene in the Hansenula polymorpha chromosome was intact and not destroyed. They all play an important role and show unique advantages of the Hansenula polymorpha expression system. A pair of primers were designed to simultaneously amplify the MOX gene (single copy) and the heterologous integrated HBsAg foreign gene (multicopy) in the Hansenula polymorpha chromosome. By comparing the brightness of the bands of the amplified product in agarose gel electrophoresis, it was possible to roughly determine whether the HBsAg gene was multiple copies. This method was used for the preliminary screening of multi-copy strains of engineered HBsAg gene. The amplified HBsAg fragment was 800 bp in length and the amplified MOX fragment was 2000 bp in length.

(24) Design Using Primer Sequences:

(25) TABLE-US-00001 primerforward: 5-TCAAAAGCGGTATGTCCTTCCACGT-3 primerreverse: 5-TACTGCTGCCAGTGCACGGTG-3

(26) PCR product agarose gel electrophoresis: the amplified product of HBsAg gene of engineering bacteria was about 800 bp, and the amplification product of Hansen yeast single copy gene MOX gene was about 2000 bp. The electrophoresis photograph of the PCR amplification product of the engineered strain obtained by screening from over 100 copies transformant was shown in FIG. 3, wherein 1 was Marker.

(27) The obtained engineering strain was tested by using 30 liters of pilot fermentation to express the HBsAg content expressed by recombinant Hansenula polymorpha in three batches of fermentation stock solution:

(28) The protein concentration was measured by the Lowry method protein quantitative kit according to national standards. The standard product was bovine serum albumin provided by the kit, and the regression curve: y=1.0368x0.0109 and R2=0.999. The fermentation stock solution was diluted 5 times with physiological saline, and the absorbance was measured. Each batch was tested twice, and the concentration of HBsAg in the corresponding pure stock solution was calculated, and the yield of HBsA was calculated as shown in the following table.

(29) TABLE-US-00002 HBsAg Fermen- HBsAg 750 nm average tation yield Absor- HBsAg concen- liquid (mg/L) batch bance concen- tration volume Fermentation number value tration (mg/mL) (mL) liquid Pure 0.306 1.491 1.527 260 397 20150606 0.312 1.563 Pure 0.253 1.257 1.270 260 330 20150617 0.258 1.283 Pure 0.276 1.376 1.395 260 363 20150624 0.281 1.402

(30) The purity of the recombinant Hansenula polymorpha HBsAg was analyzed by high pressure liquid chromatography, and the purity of the recombinant HBsAg pure stock solution was over 97%. An electron micrograph of the pure stock solution of recombinant Hansenula polymorpha recombinant HBsAg was shown in FIG. 4. The results showed that the high purity, high concentration and virus-like particle (VLP) structure of recombinant HBsAg were stable.

(31) New Product of Hepatitis B Vaccine with Recombinant Hansenula Polymorpha HBsAg for Adult was 40 g/Dose and for Children was 20 g/Dose:

(32) In 2010, the European Hepatitis B Immunology Consensus Group reported that hepatitis B surface antigen (HBsAg) immunization was the basis for anti-hepatitis B immunity. Anti-HBs were detected after inoculation to assess the intensity of the immune response in history. Although 10 mIU/ml was usually taken as a protective response, low levels of anti-HBs mask significant HBsAg infection. This led to some countries (eg, the United Kingdom, 1996) using a higher protective response of 100 mIU/ml; anti-HBs<100 mIU/ml was judged to be low/non response.

(33) On this basis, after the prophylactic hepatitis B vaccine immunization formed in recent years, the international grading standard for serological detection of protective anti-HBs response level: anti-HBs<10 mIU/ml is judged as non-response; 10 mIU/mlanti-HBs<100 mIU/ml was judged as low response; 100 mIU/mlanti-HBs<1000 mIU/ml was judged as a normal response; and anti-HBs1000 mIU/ml was judged as a high response.

(34) As mentioned above, according to the above four major hepatitis B vaccines in China, only the anti-HBs response level of the 10 g/dose newborn Hansenula polymorpha recombinant hepatitis B vaccine fully complies with the above classification criteria. The other three varieties: 5 g/dose newborn S. cerevisiae recombinant hepatitis B vaccine, 20 g/dose adult S. cerevisiae recombinant hepatitis B vaccine and 20 g/dose adult CHO recombinant hepatitis B vaccine, all had a low/non response rate of up to 30%, and the average titer GMT was low at 300 mIU/ml. In particular, there were currently no hepatitis B vaccine varieties suitable for adults in China. The market demand for recombinant hepatitis B vaccine, which was urgently needed to develop high anti-HBs immune response; it has become the driving force for Hansenula polymorpha recombinant HBsAg in adult for 40 g/dose and in children for 20 g/dose.

(35) The practice showed that the anti-HBs response level of hepatitis B vaccine is dose-dependent of HBsAg antigen, which is a common ubiquitous curve relationship. As the dose of HBsAg antigen increases gradually, the response level of Anti-HBs increases slowly. After the dose of HBsAg antigen increased to a certain point, the anti-HBs response level entered a rapidly growing linear region; in the linear region, antigen doubling induced an increase in the anti-HBs response several times; in the linear region, the HBsAg antigen dose had at least four doubling points. When the dose of HBsAg antigen increased again, the level of Anti-HBs response turned to a slow increase. Therefore, the dose of HBsAg antigen for children and adult vaccines should be selected in the corresponding linear phase of the rapid response of the response curve. If the children's HBsAg antigen dosage was 5 g/dose at the 2nd doubling point of the linear region, the HBsAg antigen dose was 10 g/dose at the 3rd doubling point; when the children's HBsAg antigen dosage was doubled to 20 g/dose at the 4th doubling point, the Anti-HBs response level still had several times the potential to increase. The above-mentioned hepatitis B vaccine anti-HBs response level and the dose of the HBsAg was dose-dependent relationship, which became a new hepatitis B vaccine product for determining the anti-HBs response rate of Hansenula polymorpha recombinant HBsAg adult dose of 40 g/dose and children dose of 20 g/dose for an important theoretical basis. The anti-HBs response of the above Hansenula polymorpha recombinant HBsAg hepatitis B vaccine product was expected to: geometric mean titer for all adults aged 15-59: GMC>Anti-HBs1000 mIU/ml, low/non response rate (<Anti-HBs 100 mIU/ml)<5%, high response rate (Anti-HBs 1000 mIU/ml)>50%; geometric mean titers of neonates with high prevalence of HBsAg and older children aged 10-13: GMC>Anti-HBs 2000 mIU/ml, low/non response rate <5%, and high response rate >60%.

(36) The yield of HBsAg pure stock solution of the recombinant Hansenula polymorpha provided by the present invention reached 300 mg/liter or more, and the yield of the purified HBsAg pure stock solution of the existing Hansenula polymorpha or brewer's yeast was much higher than 5 times to 30 times; the highest level in the world today, a new breakthrough in genetic recombination technology. The annual production of recombinant HBsAg pure stock solution was calculated on the scale of two fermenters with 800 liter:
2 products (raw liquid liter (working volume)(working volume) liter (work tank/year)=19,800 g/year

(37) It was a prerequisite for the large-scale production of hepatitis B vaccine with Hansenula polymorpha recombinant HBsAg adult dose of 40 g/dose and children dose of 20 g/dose. When the annual yield of Hansenula polymorpha recombinant HBsAg VLP pure stock solution reaches 19,800 grams, the dosage of Hansenula polymorpha recombinant HBsAg for adult was 40 g/dose and the annual output of hepatitis B vaccine was 330 million needles (110 million people); the dosage of Hansenula polymorpha recombinant HBsAg for children was 20 g/dose and the annual output of hepatitis B vaccine was 330 million needles (110 million people); it will become the largest manufacturer of recombinant hepatitis B vaccine in the world for meet the demand of expansion of immunization for all populations in the country.

First Embodiment

(38) The pMPT-HBsAg adw2 plasmid was constructed based on the sequence of SEQ ID NO: 1. The construction of plasmid pMPT-HBs adw2 includes the following steps:

(39) The HBsAg adw2 gene was synthesized according to the DNA sequence of Hansenula polymorpha preferred code HBsAg adw2; and the glycerol strain containing the HBsAg adw2 gene plasmid was constructed and named as MC407B-16.

(40) The correctly sequenced MC407B-16 plasmid was digested with EcoRI/BamHI, and the digested product was used a TaKaRa PCR Fragment Recovery Kit (Code No. D301) to recover 701 bp DNA fragment called Inset DNA6.

(41) The correct plasmid pMPT-02 was digested with EcoRI/BamHI, and the vector DNA obtained after the gel was recovered was called Vector DNA6.

(42) Inset DNA6 was ligated to Vector DNA6 using Solution in TaKaRa DNA Ligation Kit (Code No. D6022), and then heat-shock transformed into E. coli Competent Cell JM109 (Code No. D9052), and the cells were cultured overnight.

(43) Single colonies were selected from the transformation plates, and plasmid DNA was extracted and digested with EcoRI/BamHI. The results showed that MC407A+B+C+D-7780 were positive clones.

(44) The plasmid MC407A+B+C+D-77 was sequenced respectively with primers RV-M, M13-47, MC407P1, MC407P2, MC407P3, MC407P4, MC407P5, MC407P6, MC407P7, MC407P8, MC407P9, MC407BF11, MC407BR11 to prove the plasmid pMPT-HBs adw2 were correct.

Second Embodiment

(45) Construction of a Hansenula polymorpha recombinant HBsAg engineering strain (i.e., a Hansenula polymorpha host cell transformation screening strain comprising the sequence set forth in SEQ ID NO: 1).

(46) The transformation and screening process of recombinant Hansenula polymorpha hepatitis B vaccine was shown in FIG. 5:

(47) 1) The pMPT-HBsAg plasmid was transformed into the URA3-auxotrophic Hansenula polymorpha cell strain HU-11 (CGMCC No. 1218) of the host cell by cell electroporation. The culture medium was selected using a selection medium (MD liquid medium). The single colony transformants were picked up on the MD selection culture plate and transferred to the MD liquid medium for continuous subculture. The adw2 subtype HBsAg gene and the corresponding regulatory components were multi-copy and heterologously integrated into the host Hansenula polymorpha cell chromosome.

(48) 2) Strain screening included the following steps:

(49) (1) Selecting a single colony of uracil prototrophic transformants

(50) Colonies with rapid growth rate of bacteria were selected. PCR was used to detect the brightness of HBsAg gene bands. Colonies with a large number of copies were selected, and single colonies were shake-cultured in a selective medium, and successively subcultured for 20 to 400 generations;

(51) (2) Screening multiple copies of heterologous integrated transformed clones

(52) After subculture in step (1), after 72 hours of methanol-induced culture, the expression level of HBsAg released by the disruption of transformant cells was determined by radio immunoassay or radioimmunoassay (RIA);

(53) (3) Screening out high-copy, high-expression clones of free plasmids

(54) The clones screened by step (2) were cultured in YPD complete medium for 48 hours, and then transferred into a selection medium plate for cloning culture, and the HBsAg gene copy number was detected by quantitative PCR, and the expression level of HBsAg was detected by RIA.

(55) (4) Based on the detection result of the step (3), the primary strain of the genetically stabilized recombinant Hansenula polymorpha HBsAg engineering strain was selected.

Third Embodiment

(56) The main process of 30 liters of pilot fermentation:

(57) 1) Strain stored in liquid nitrogen was thawed by 200 ml seed medium, inoculated into the medium, divided into two 0.5 L shake flasks, and cultured at 31 C. for 22 hours as a first-class seed;

(58) 2) The primary seed was transferred into the secondary seed culture medium with 1600 ml seed medium, divided into six 1 L shake flasks, and incubate at 31 C. for 20 hours as a secondary seed;

(59) 3) 12 L fermentation medium was adjusted to pH 5.5 and transferred into a 30 L fermenter, and then the secondary seed was inoculated under growing at 30-31 C. through two sources of glycerol and methanol; growth, de-repression and induction for the three phases, and co-culture 85 to 96 hours, the cells were harvested after 2-3 hours stopped induction. The frozen cells are homogenized.

(60) Operation Points:

(61) (1) The feeding operation of the growth phase was going when the dissolved oxygen was consumed and the basal medium was consumed; the flow acceleration was gradually increased as the consumption of the basic medium increases, and the flow was added before 2-3 hours the dissolved oxygen was recovered.

(62) (2) In the later stage of the growth phase, pay attention to the dissolved oxygen recovery, record the lowest value of dissolved oxygen, and start to flow when the dissolved oxygen rises to 70-80%, and enter the de-repression phase.

(63) (3) After the later stage of the de-repression phase, the dissolved oxygen began to rise after the end of the flow. When the dissolved oxygen was raised to 70-80% c/o, the methanol induction solution was added, and the methanol concentration is controlled at 3-5; the flow acceleration was controlled by the methanol detection flow controller.

(64) (4) Stopping methanol addition before 2-3 hours the end of fermentation to reduce methanol residue during cell harvest.

(65) Medium

(66) 1. Preparation of calcium chloride solution

(67) 11.33 g CaCl.sub.2 was accurately weighed and put it into a cleaned triangular flask, deionized water was appropriately added to dissolve and dilute to 200 ml.

(68) 2. Preparation of Micro Element Solution

(69) Accurately weighting the following reagents:

(70) TABLE-US-00003 (NH.sub.4).sub.2Fe(SO.sub.4).sub.2e(.sub.2O 1000 mg CuSO.sub.4.sub.2O 80 mg ZnSO.sub.4.sub.2O 300 mg MnSO.sub.4.sub.2O 400 mg EDTA 1000 mg

(71) The weighed reagent was placed in a cleaned triangular flask, dissolved in deionized water and dissolved to 200 ml.

(72) 3. Preparation of Vitamin Solution

(73) Accurately weighting the following reagents:

(74) TABLE-US-00004 d-Biotin 6 mg Thiamin HCl 2000 mg

(75) Biotin was first dissolved in 10 ml of 50% isopropanol, and then dissolved in Thiamin HCl, and then dissolved in an appropriate amount of deionized water to a volume of 100 ml.

(76) 4. Preparation of Trace Element Solution

(77) Accurately weighting the following reagents:

(78) TABLE-US-00005 NiSO.sub.4.sub.2O 10 mg CoCl.sub.2.sub.2O 10 mg H.sub.3BO.sub.3 10 mg Na.sub.2MoO.sub.4oO.sub.2O 10 mg KI 10 mg

(79) The weighed reagent was placed in a cleaned triangular flask, and an appropriate amount of deionized solution was added to a volume of 50 ml.

(80) The above four solutions were separately sterilized and filtered for use.

(81) 5. Preparation of Seed Salt Solution

(82) Accurately weighting the following reagents:

(83) TABLE-US-00006 NH.sub.4H.sub.2PO.sub.4 80 g MgSO.sub.4gS.sub.2O 18 g KCl 20 g NaCl 2 g

(84) The weighed reagent was placed in a cleaned triangular flask, dissolved in deionized water and dissolved to a volume of 1600 ml.

(85) 6. 27 g of glycerin was weighted in a 2000 ml flask, mixed with a salt solution of 360 mL, and made up to 1800 ml with deionized water. The same amount was dispensed into two 2000 ml flasks, and autoclaved at 110 C. for 30 minutes.

(86) Two empty 500 ml triangle bottles, six 1000 ml triangle bottles, a 100 ml graduated cylinder and a 500 ml graduated cylinder all were sterilized under 110 C., 30 minutes high pressure steam.

(87) 7. Primary Seed Medium

(88) In the clean bench, 100 ml of each sterilized glycerin solution was taken aseptically, and added separately into two 500 ml sterilized flasks, and respectively added the following:

(89) TABLE-US-00007 Calcium chloride solution 1 ml Micro element solution 1 ml Vitamin solution 0.5 ml Trace element solution 0.25 ml Shaking the above solution.

(90) 8. Secondary Seed Medium

(91) 1600 ml of sterilized glycerin solution was placed in a clean bench with sterile operation technique and placed in a 2000 ml sterilized triangle, and separately added:

(92) TABLE-US-00008 Calcium chloride solution 16 ml Micro element solution 16 ml Vitamin solution 8 ml Trace element solution 4 ml

(93) 9. Fermentation Medium

(94) The following reagents were accurately weighted and dissolved in 2000 ml of deionized water.

(95) TABLE-US-00009 NH.sub.4H.sub.2PO.sub.4 175 g MgSO.sub.4gS.sub.2O 40 g KCl 44 g NaCl 4.4 g

(96) 520 g glycerin was weighted and added into a small 500 ml beaker. 10 ml defoamer was added into the beaker to sterilize, and then added:

(97) TABLE-US-00010 Calcium chloride solution 175 ml Micro element solution 175 ml Vitamin solution 88 ml Trace element solution 44 ml

(98) 10. Feed Medium

(99) 87 g NH.sub.4H.sub.2PO.sub.4, 260 g glycerin and 500 ml deionized water were added into 1000 ml flask, and then wrapped feed line and sterilized at 110 C. for 30 minutes.

(100) 11. De-Repression Solution

(101) 1800 g glycerin and 660 ml deionized water were added into a 5000 ml flask, and then wrapped feed line and sterilized at 110 C. for 30 minutes. 540 ml filter-sterilized salt solution was added after cooling.

(102) 12. Induction Solution

(103) 400 ml glycerin was added into a 1000 ml flask, and then wrapped feed line and sterilized at 110 C. for 30 minutes. 1600 ml methanol was added aseptically after cooling.

Fourth Embodiment

(104) Purification

(105) The fermentation broth obtained from the third embodiment was harvested and the cells were washed. The detailed steps of purification can be found in References: Li Jin, Kong Yan. Recombinant Hepatitis B Vaccine Production Process. See Li Jin, Yu Yu, Dong Dexiang Editor: Biopharmaceutical Equipment And separation and purification techniques. 1st edition. Beijing: Chemical Industry Press, 2003: 348-349. The harvested cells can be crushed by a homogenizer to release HBsAg; the cell debris was removed by filtration with a 0.22 g microporous filter; the small molecular impurities were removed by ultrafiltration with a 300K ultramicrofilter; and the HBsAg was extracted by silica gel adsorption treatment. Finally, it was purified by butyl agarose hydrophobic chromatography.

REFERENCES

(106) 1, Qi Xiaoqiu, etc., the national population of hepatitis B virus epidemiology investigation report, the first edition of April 2011, People's Health Publishing House. 2, Zhuang Hui, China's hepatitis B prevention and treatment status and goals, 2008, meeting. 3. Li Jian et al. Low/no response study after neonatal vaccination with recombinant hepatitis B vaccine (yeast) in Shanghai, China Vaccine and Immunity, 2011, Vol. 17 No. 5: 399-403. 4. Liu Jiaye et al. Comparative study on the immune response and its influencing factors of 20 g recombinant hepatitis B vaccine in adults, Chinese Journal of Vaccines and Immunity, 2013, Vol. 19, No. 2: 142-146. 5. European Consensus Group on Hepatitis B Immunity. Are booster immunisations needed for lifelong hepatitis B immunity? Lancet 2000; 355: 561-565.

(107) The foregoing description of the exemplary embodiments of the disclosure has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.

(108) The embodiments were chosen and described in order to explain the principles of the disclosure and their practical application so as to enable others skilled in the art to utilize the disclosure and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present disclosure pertains without departing from its spirit and scope.