CN118109443A

CN118109443A - Recombinant protein antigen of oral helicobacter pylori vaccine, preparation and application

Info

Publication number: CN118109443A
Application number: CN202410532695.1A
Authority: CN
Inventors: 陈克平; 陈正军; 刘冬; 李建霖; 樊钒; 税静; 朱冲; 江之永; 陶杨; 明辉
Original assignee: Chengdu Olymvax Biopharmaceuticals Inc
Current assignee: Chengdu Olymvax Biopharmaceuticals Inc
Priority date: 2024-04-30
Filing date: 2024-04-30
Publication date: 2024-05-31

Abstract

The invention relates to the biomedical field, in particular to an oral helicobacter pylori vaccine recombinant protein antigen, and preparation and application thereof. In the prior art, the purification difficulty of the UreB protein is high, and the oral recombinant protein vaccine tolerance and antigen are diluted, degraded and even denatured and inactivated. The invention provides an oral helicobacter pylori vaccine recombinant protein antigen UreB and a preparation method thereof, histidine (his) and glutathione transferase (GST) proteins are added on the UreB protein, the recombinant protein antigen UreB is expressed as his-GST-UreB fusion protein, the purity of the UreB protein reaches 95% after purification, and the yield of each liter of fermentation broth can be obtained to 122mg of protein. Animal experiments prove that the vaccine can effectively stimulate organisms to generate immune response and has good immune protection effect, and can be used as a vaccine candidate component for preventing helicobacter pylori infection.

Description

Recombinant protein antigen of oral helicobacter pylori vaccine, preparation and application

Technical Field

The invention relates to the biomedical field, in particular to an oral helicobacter pylori vaccine recombinant protein antigen, and preparation and application thereof.

Background

Helicobacter pylori (Helicobacter pylori, hp) is a gram-negative, helicobacter, microaerophilic bacterium that persists in the stomach, mucosa and duodenal epithelium. 1982. Marshall and Warren, the annual Australian scholars, reported for the first time that the separation of helicobacter pylori from the gastric mucosa of patients with chronic gastritis and gastric ulcers, is the only species of microorganism currently known to survive in the human stomach. Numerous studies have demonstrated that Hp is a common causative agent of gastrointestinal disease, and is the primary causative agent of chronic type B gastritis, gastric and duodenal ulcers. Hp is also a key cause of gastrointestinal cancer and gastric mucosa lymphoid tissue lymphoma, and genetic instability of gastric epithelial cells is caused by regulating and controlling intracellular signals, so that a DNA damage repair system is influenced, and tumor transformation of the gastric epithelial cells is promoted. The international cancer research center in 1994 listed it as the primary class I cancer carcinogen. 2017. The list of carcinogens published by the international cancer research institute of the world health organization shows that helicobacter pylori belongs to class I carcinogens and can induce tumors such as gastric cancer, lymphoproliferative gastric lymphoma and the like.

Urease (Urase) is a nickel-dependent enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid, and is the most abundant protein expressed by helicobacter pylori in cells and on cell membranes, accounting for 5% -10% of total Hp protein. Urease breaks down urea to produce ammonia, aids in bacterial colonization in the stomach by neutralizing gastric acid, and provides ammonia for bacterial protein synthesis. Urease is distributed on the surface and inside of bacteria and is purchased from two subunits, ureA and UreB. In the aspect of the pathogenic mechanism of HP, urease mainly hydrolyzes urea to produce ammonia and carbon dioxide, and the pH value around bacterial cells is regulated, so that the HP is key to survival and colonization in high-strength gastric acid, and meanwhile, urease is an important colonization factor and virulence factor of HP. The HP strains in different areas have the characteristics of diversity, easy variation and the like, but the nucleic acid sequences of UreB among different HP strains have high conservation and homology, have large molecular weight and strong specificity, can effectively induce organisms to generate stable immunoprotection, and compared with UreA, the UreB has better immunoprotection. Thus, ureB is listed as one of the most promising candidate antigens. Urease B antibodies can be detected in all patients infected with Hp, especially those with overt symptoms, and the antibody levels are related to some extent to the severity of the disease condition.

The human mucosal immune system (Human mucosal immune system, HMIS) is mainly composed of mucosa and submucosal lymphocytes which are dispersed and distributed in the respiratory tract, genitourinary tract, digestive tract and the like, is the largest organ of the human body, and the surface area of the mucosa and submucosal lymphocytes is larger than that of the skin. Wherein, intestinal mucosa is the most main mucosa part of human body. The human intestinal tract has a surface area of about 400 cm ². In the intestinal mucosal system, there are unique lymphoid tissues and other large numbers of lymphocytes that recognize and resist the foreign dangerous pathogens through their innate and acquired immunity, thereby maintaining the immune homeostasis of the body. At the mucosal site of the intestinal tract, the uptake and presentation of antigen by immune cells can induce both systemic humoral and cellular immune responses, such as the production of antibodies (mainly sIgA) and corresponding cytotoxic T Cells (CTL) against the antigen, due to the abundance of immune cells. Meanwhile, activated antigen-specific lymphocytes home to remote mucous membrane effect sites and can induce local immune responses of mucous membranes. Thus intestinal mucosal immunity has great potential to achieve systemic or local immunotherapy. Intestinal mucosal vaccination is also currently considered the only way to defend against enteropathogenic bacteria.

Bacterial DNA is a natural ligand for Toll-like receptor 9 and synthetic Oligodeoxynucleotides (ODNs) containing unmethylated CpG sequences can mimic bacterial DNA structure and elicit similar activity. CpG ODNs can trigger TLR9 expressing immune cells (including human plasmacytoid dendritic cells and B cells) to produce innate immune responses characterized by Th1 immunity and its associated cytokines. CpG ODNs, when used as vaccine adjuvants, can enhance the function of professional antigen presenting cells and promote antigen-specific humoral and cellular immune responses. CpG-containing sequences can be divided into three major classes, depending on structure and biological function: ext> CpGext> -ext> Aext>,ext> CpGext> -ext> Bext> andext> CpGext> -ext> Cext> classesext>,ext> withext> CpGext> -ext> Bext> classext> moleculesext> beingext> theext> mostext> commonlyext> usedext> inext> clinicalext> vaccineext> researchext>.ext> The structure and function of CpG-ODN are closely related. In fact, the higher structure of the molecule determines whether CpG-ODNs localize in the cell early or late lysosomes, which are associated with different signaling pathways. Ext> multimericext> CpGext> -ext> Aext> ODNsext> areext> mainlyext> localizedext> toext> earlyext> lysosomesext>,ext> inext> plasmacytoidext> dendriticext> cellsext>,ext> whichext> leadext> toext> strongext> inductionext> ofext> IFNext> -ext> αext>.ext> The monomeric CpG-B ODNs concentrate in late endosomal compartments and can promote cell maturation of plasmacytoid dendritic cells and B cells. CpG-C ODNs localize to two compartments, inducing IFN- α production and cell maturation. Other structural modifications that affect the biological effects of CpG-containing sequences include linking two or more short phosphorothioate backbone CpG ODNs via non-nucleoside chemical linkers to produce formulations of linear chimeric immunomodulatory compounds and/or CpG nanoparticle-containing compounds. 1018 is a synthetic CpG-B class oligonucleotide having a phosphorothioate backbone and sequence 5'-TGACTGTGAACGTTCG AGATGA-3'. The first CpG motif (underlined) is a sequence active on mouse TLR-9, while the second CpG motif (underlined) is active on human and non-human primate TLR-9. CpG 1018 has been used as an adjuvant for Heplisav-B, a modified HBV vaccine, for adults (age >18 years). Although other HBV vaccines have been widely used, they are typically administered in a three dose regimen, whereas Heplisav-B provides a two dose regimen.

Disclosure of Invention

The invention aims to provide an oral helicobacter pylori vaccine recombinant protein antigen and preparation and application thereof, which solve the technical problems of great difficulty in purifying UreB protein, dilution, degradation and even denaturation inactivation of the oral recombinant protein vaccine tolerance and antigen in the prior art.

In order to achieve the above purpose, the present invention provides the following technical solutions:

The invention provides an oral helicobacter pylori vaccine recombinant protein antigen, which is UreB, and has an amino acid sequence shown in SEQ ID NO:1, the amino acid sequence of the fusion protein his-GST-UreB is shown as SEQ ID NO:2, the encoding nucleotide sequence of the fusion protein his-GST-UreB is shown as SEQ ID NO. 3.

The invention provides a preparation method of an oral helicobacter pylori vaccine recombinant protein antigen, which comprises the following steps:

S1, constructing a plasmid, and expressing protein: linking a his-GST-UreB gene with a nucleotide sequence shown as SEQ ID NO. 3 in an expression vector, and transferring the constructed expression vector into recombinant engineering bacteria for induced expression;

s2, fermenting;

S3, purifying;

S31, re-suspending the strain; adding the thallus and the solution A into the solution A according to the mass ratio of 1:10 for dilution and resuspension until the thallus is uniformly resuspended in the solution A;

s32, crushing thalli, centrifugally filtering and collecting supernatant;

S33, ni-FF1 chromatography: collecting a sample by adopting the steps of liquid A balance, sample loading, liquid A re-balance, 7.5% liquid B impurity washing and 30% liquid B elution;

S34, G25 liquid exchange: collecting a sample by adopting the steps of liquid C balancing, sample loading and liquid C eluting;

s35, enzyme cutting: adding the ppase into the protein and the ppase according to the ratio of v/v=20:1, slightly stirring and mixing uniformly, and standing at 2-8 ℃ for overnight digestion; filtering, sampling and detecting;

s36, ni-FF2 chromatography, namely collecting a sample by adopting the steps of C liquid balance, sample loading, C liquid re-balance, 4%D liquid elution and 50% D liquid impurity washing;

S37, Q HP chromatography, balance: the balance of the solution F is 1-2CV, and the balance of the solution E is 1-5CV; loading a sample; re-balancing 2-3CV;0-100% F solution, and collecting eluting peak after 10CV elution;

S8, ultrafiltration liquid exchange, sterilization and filtration are carried out to obtain UreB stock solution.

Furthermore, the expression vector in the S1 is pET-29b (+), and the recombinant engineering bacterium is pET-UreB/BL21.

Further, the step S2 includes the following substeps:

S21, starting strains: the fourth generation of pET 29-UreB/BL 21 seed bacteria is represented by 1: inoculating 600v/v ratio into a triangular flask, culturing in LB culture medium at 36.0-38.0deg.C at 200-240rpm for 4-8 hr to obtain first generation strain;

S22, seed tank culture: inoculating the first generation strain in seed tank according to the inoculation amount ratio of 0.5-2.0%, filling LB culture medium in the tank, and deep ventilating: culturing for 3.0-8.0h at 10.0-50.0L/min, 36.0-38.0 ℃ and 50-300 rpm and pH 6.70-7.30 to obtain second-generation production strains;

S23, culturing in a fermentation tank: inoculating the second-generation production strain into a fermentation tank according to the inoculum size of 8% -12%, and culturing for 3.0-7.0 hours at 36.0-38.0 ℃ and deep ventilation of 50.0-350.0L/min, 100-500 rpm and pH of 6.70-7.30, wherein strain amplification is finished;

s24, induction culture: temperature: 29.0 to 31.0 ℃; deep ventilation: 50.0 to 350.0L/min; rotational speed: 100-500 rpm parts; pH: 6.70-7.30; induction time 4.0 hours;

s25, centrifugally collecting bacterial precipitate.

Further, taking bacterial liquid samples after the S21, S22 and S24 are finished, performing pure bacterial examination and microscopic examination, wherein bacteria are gram-negative bacilli, and the OD ₆₀₀ value of the bacterial liquid after the S21, S22, S23 and S25 are finished is respectively more than 1.50, 20.00 and not more than 1.00; the S23 bacterial liquid OD ₆₀₀ starts to supplement glycerol at 0.015L/min at 10.00 until the induction is finished; the S24 induction was started by adding 0.02L/min of a medium containing 24.0 g/L of yeast extract and 12.0 g/L of tryptone.

Further, the step S32 specifically includes: the resuspended cells were subjected to shear homogenization: homogenizing at 2-8 ℃ for 3 times, wherein the temperature is 700-900 Bar; centrifuging the homogenized liquid under a centrifugal force of 12000g for 30min; collecting supernatant, clarifying and filtering with 0.6-0.8 μm deep layer filter plate, and clarifying and filtering with 0.65 μm filter core.

Further, the balance in S33 is 2-3CV; loading a sample; re-balancing 3-5CV; washing: 7.5% B,2-5CV; eluting: 30% B,2-5CV; the solution A is 50mM Tris+ 0.15M NaCl+0.5%Tween-80, pH 8.0, and the solution B is 50mM Tris+0.15M NaCl+0.5M imidazole+0.5% Tween-80, pH 8.0.

Further, the solution C is 50mM Tris+0.15M NaCl, the pH is 8.0, and the balance in the step S36 is 2-3CV; loading a sample; re-balancing 2-5CV; elution conditions: 4%D, 20mM imidazole, 3-5CV; washing: 50% D solution, 250mM imidazole, 1-3CV, which is 50mM Tris+0.15M NaCl+500mM imidazole, pH8.0.

Further, the E solution in the S37 is 20mM Tris+1mM DTT,pH 7.5,F solution which is 20mM Tris+0.5M L-Arg+1mM DTT; the ultrafiltration liquid exchange in the step S38 is to carry out ultrafiltration concentration treatment on the three-step chromatographic sample liquid by using a 30KD membrane package, concentrate the three-step chromatographic sample liquid to 1/5-1 times of the original volume, supplement the G liquid with the same volume, continuously wash and filter 5-10 volumes, and concentrate the sample to 1/2-2 times of the volume of the three-step chromatographic sample liquid after dialysis; the G solution is 20mM PB+5 mM L-Cys Hcl+5% mannitol, and the pH value is 8.0.

The invention also provides an application of the recombinant protein antigen UreB of the oral helicobacter pylori vaccine in the medicine for preventing or treating helicobacter pylori infection.

Further, the medicament is a vaccine.

Based on the technical scheme, the embodiment of the invention at least has the following technical effects:

(1) According to the recombinant protein antigen UreB provided by the invention, histidine (his) and glutathione transferase (GST) proteins are added on the UreB protein, the recombinant protein antigen UreB is expressed as a his-GST-UreB fusion protein, the purity of the UreB protein reaches 95% after purification, and 122mg of protein can be obtained in the yield of each liter of fermentation broth.

(2) The toxin-attacking protection rate of the recombinant protein antigen UreB provided by the invention is 30% when the UreB is singly used, and the toxin-attacking protection rate of the UreB+CpG mice is 60%.

Drawings

FIG. 1 shows the result of the cleavage and identification of recombinant plasmid pET 29-UreB; the left side of the electrophoresis chart in fig. 1, and the right side of the electrophoresis chart in fig. 1 is a Marker;

FIG. 2 is an electrophoretogram of a his-GST-UreB fusion protein according to an embodiment of the present invention;

FIG. 3 shows the statistics of the IgG titers of serum-specific antibodies of the examples of this invention.

Detailed Description

The drawings in the embodiments of the present invention will be combined; the technical scheme in the embodiment of the invention is clearly and completely described; it is apparent that the described embodiments are only a few of the embodiments of the present invention; but not all embodiments, are based on embodiments in the present invention; all other embodiments obtained by those skilled in the art without making any inventive effort are within the scope of the present invention.

SEQ ID NO:1

1 MKKISRKEYV SMYGPTTGDK VRLGDTDLIA EVEHDYTIYG EELKFGGGKT LREGMSQSNN

61 PSKEELDLII TNALIVDYTG IYKADIGIKD GKIAGIGKGG NKDMQDGVKN NLSVGPATEA

121 LAGEGLIVTA GGIDTHIHFI SPQQIPTAFA SGVTTMIGGG TGPADGTNAT TITPGRRNLR

181 WMLRAAEEYS MNLGFLAKGN ASNDASLADQ IEAGAIGFKI HEDWGTTPSA INHALDVADE4

241 YDVQVAIHTD TLNEAGCVED TMAAIAGRTM HTFHTEGAGG GHAPDIIKVA GEHNILPAST4

301 NPTIPFTVNT EAEHMDMLMV CHHLDKSIKE DVQFADSRIR PQTIAAEDTL HDMGIFSITS4

361 SDSQAMGRVG EVITRTWQTA DKNKKEFGRL KEEKGDNDNF RIKRYLSKYT INPAIAHGIS4

421 EYVGSVEVGK VADLVLWSPA FFGVKPNMII KGGFIALSQM GDANASIPTP QPVYYREMFA

481 HHGKAKYDAN ITFVSQAAYD KGIKEELGLE RQVLPVKNCR NITKKDMQFN DTTAHI EVNPI

541 ETYHVFVDGK EVTSKPATKV SLAQLFSIFY

SEQ ID NO:2

MGSSHHHHHH SSGSPILGYW KIKGLVQPTR LLLEYLEEKY EEHLYERDEG DKWRNKKFEL

GLEFPNLPYY IDGDVKLTQS MAIIRYIADK HNMLGGCPKE RAEISMLEGA VLDIRYGVSR

IAYSKDEETL KVDFLSKLPE MLKMFEDRLC HKTYLNGDHV THPDEMLYDA LDVVLYMDPM

CLDAFPKLVC FKKRIEAIPQ IDKYLKSSKY IANPLQGWQA TFGGGDHPPK SDLEVLFQGP

KKISRKEYVS MYGPTTGDKV RLGDTDLIAE VEHDYTIYGE ELKFGGGKTL REGMSQSNNP

SKEELDLIIT NALIVDYTGI YKADIGIKDG KIAGI GKGGN KDMQDGVKNN LSVGPATEAL

AGEGLIVTAG GIDTHIHFIS PQQIPTAFAS GVTTMIGGGT GPADGTNATT IT PGRRNLKW

MLRAAEEYSM NLGFLAKGNA SNDASLADQI EAGAIGFKIH EDWGTTPSAI NHALDVADKY

DVQVAIHTDT LNEAGCVEDT MAAIAGRTMH TFHTEGAGGG HAPDIIKVAG EHNILPASTN

PTIPFTVNTE AEHMDMLMVC HHLDKSIKED VQFADSRIRP QT IAAEDTLH DMGI FSITSS

DSQAMGRVGE VITRTWQTAD KNKKEFGRLK EEKGDNDNFR IKRYLSKYTI NPAIAHGISE

YVGSVEVGKV ADLVLWSPAF FGVKPNMIIK GGFIALSQMG DANASIPTPQ PVYYREMFAH

HGKAKYDANI TFVSQAAYDK GIKEELGLER QVLPVKNCRN ITKKDMQFND TTAHIEVNPE

TYHVEVDGKE VTSKPATKVS LAQLFSIF

SEQ ID NO:3

CATATGGGCT CTAGCCATCA CCACCACCAC CACTCTAGCG GTAGCCCGAT CCTGGGCTAC

TGGAAAATCAA AGGTCTGGT TCAGCCGACC CGTCTGCTGC TGGAATACCT GGAAGAAAAA

TACGAAGAAC ACCTGTACGA ACGTGATGAA GGTGATAAAT GGCGTAACAA AAAATTCGAA

CTGGGTCTGG AATTCCCGAA CCTGCCGTAC TACATCGATG GCGATGTTAA ACTGACCCAG

AGCATGGCGA TCATCCGTTA CATCGCGGAT AAACACAACA TGCTGGGCGG CTGCCCGAAA

GAACGTGCGG AAATCAGCAT GCTGGAAGGC GCTGTTCTGG ATATCCGTTA CGGTGTTTCT

CGTATCGCGT ACTCTAAAGA TTTCGAAACC CTGAAAGTTG ATTTCCTGAG CAAACTGCCG

GAAATGCTGA AAATGTTCGA AGATCGTCTG TGCCACAAAA CCTACCTGAA CGGCGATCAC

GTTACCCACC CGGATTTCAT GCTGTACGAT GCGCTGGATG TTGTTCTGTA CATGGACCCG

ATGTGCCTGG ATGCGTTCCC GAAACTGGTT TGCTTCAAAA AACGTATCGA AGCGATCCCG

CAGATCGATA AATACCTGAA ATCTAGCAAA TACATCGCGT GGCCGCTGCA GGGTTGGCAG

GCGACCTTCG GCGGTGGTGA TCACCCGCCG AAAAGCGATC TGGAAGTTCT GTTCCAGGGC

CCGAAGAAAA TCTCTCGTAA AGAATACGTT AGCATGTACG GCCCGACCAC CGGTGATAAA

GTTCGTCTGG GTGATACCGA TCTGATTGCT GAAGTTGAAC ACGATTACAC CATCTACGGT

GAAGAACTGA AATTCGGCGG TGGTAAAACC CTGCGTGAAG GTATGAGCCA GTCTAACAAC

CCGAGCAAAG AAGAACTGGA TCTGATCATC ACCAACGCAC TGATCGTTGA TTACACCGGC

ATTTATAAAG CAGATATCGG TATTAAAGAT GGTAAAATCG CGGGCATCGG CAAAGGTGGC

AACAAAGATA TGCAGGATGG TGTTAAAAAC AACCTGTCTG TTGGCCCGGC GACCGAAGCG

CTGGCGGGTG AAGGCCTGAT CGTTACCGCA GGTGGCATTG ATACCCATAT CCACTTCATT

TCTCCGCAGC AGATCCCGAC CGCTTTCGCG AGCGGTGTTA CCACCATGAT CGGCGGTGGC

ACCGCCCGG CGGATGGCAC TAACGCGACC ACCATCACCC CTGGCCGTCG CAACCTGAAA

TGGATGCTGC GCGCTGCAGA AGAATACAGC ATGAACCTGG GCTTCCTGGC TAAAGGCAAC

GCTTCTAACG ATGCGTCCCT GGCGGATCAG ATCGAAGCTG GCGCGATCGG TTTCAAAATC

CACGAAGATT GGGGCACCAC CCCGAGCGCT ATCAACCACG CACTGGATGT TGCAGATAAA

TACGATGTGC AGGTTGCTAT TCACACCGAT ACCCTGAACG AAGCGGGCTG CGTTGAAGAT

ACCATGGCTG CGATTGCAGG CCGTACCATG CACACCTTTC ATACCGAAGG CGCAGGTGGC

GGCCACGCGC CGGACATCAT CAAAGTTGCT GGCGAACACA ACATCCTGCC GGCTAGCACC

AACCCGACCA TCCCGTTCAC CGTTAACACC GAAGCAGAAC ACATGGATAT GCTGATGGTT

TGCCACCACC TGGACAAATC CATTAAAGAA GATGTTCAGT TCGCGGATAG CCGTATCCGT

CCGCAGACCA TTGCGGCTGA AGATACTCTG CACGACATGG GCATCTTCTC TATCACCTCC

TCCGATAGCC AGGCGAIGGG CCGTGTGGGC GAAGTTATCA CCCGCACCIG GCAGACCGCT

GATAAAAACA AAAAAGAATT CGGTCGTCTG AAAGAAGAAA AAGGTGATAA CGACAACTTC

CGTATCAAAC GTTACCTGTC TAAATACACC ATCAACCCGG CTATTGCTCA CGGTATCAGC

GAATATGTGG GTAGCGTGGA AGTTGGCAAA GTTGCGGATC TGGTTCTGTG GTCCCCGGCA

TTCTTCGGCG TGAAACCGAA CATGATCATC AAAGGCGGCT TTATCGCACT GTCCCAGATG

GGTGACGCGA ACGCGAGCAT CCCGACCCCG CAGCCGGTTT ACTACCGTGA AATGTTTGCG

CACCACGGCA AAGCGAAATA CGATGCTAAC ATCACCTTCG TTAGCCAGGC GGCTTATGAT

AAAGGCATTA AAGAAGAACT GGGTCTGGAA CGTCAGGTTC TGCCGGTTAA AAACTGCCGT

AACATTACCA AAAAAGATAT GCAGTTCAAC GACACCACCG CGCACATCGA AGTTAACCCG

GAAACCTACC ACGTTTTCGT TGACGGTAAA GAAGTTACCT CTAAACCGGC GACCAAAGTT

AGCCTGGCGC AGCTGTTCTC TATCTTCTAA GGATCC

Example 1

S1, constructing a plasmid, and expressing protein: the his-GST-UreB gene with the nucleotide sequence shown as SEQ ID NO. 3 is linked in an expression vector, and the constructed expression vector is transferred into recombinant engineering bacteria for induced expression.

The G272-WP-000724296.1 is adopted as a UreB sequence, the UreB protein is not easy to purify, 6 histidine (his) and glutathione S-transferase (GST) proteins are added during construction, so that the UreB protein is expressed as a his-GST-UreB fusion protein, and the fusion protein is separated and purified through glutathione agarose affinity chromatography specifically combined with GST, wherein the his-GST-UreB fusion protein sequence is shown as SEQ ID NO. 2.

The target gene is synthesized by Shanghai biological engineering Co., ltd, as shown in figure 1, recombinant plasmid pET29-UreB (his-GST-UreB) is digested to obtain 5.3kb vector fragment and 2436bp target fragment, which are consistent with the expected result, and the recombinant plasmid is constructed correctly.

The engineering strain is BL21 (DE 3), the genotype is F-ompT hsdSB (rB-mB-) gal dcm (DE 3), and the engineering strain is derived from Shanghai biological engineering Co. The invention entrusts Shanghai biological engineering limited company to transform a recombinant expression vector pET29b (+) -UreB into host bacterium BL21 (DE 3).

The induction expression is specifically as follows:

pET 29-UreB/BL 21 seed bacteria were inoculated into 10ml LB medium and cultured in shaking table at 37℃for 12-16h with 220 rpm. 400 μl of the culture bacteria liquid is taken and added into 20ml of kana-resistance LB liquid culture medium, and the culture is put into a shaking table at 37 ℃ and cultured at 220rpm until the OD600 is 0.6-0.8. The bacterial liquid was collected and centrifuged at 10000rpm for 10min at 1ml, the supernatant was removed, and the bacterial liquid was stored at-20℃and examined as an uninduced sample. IPTG with a final concentration of 0.5mmol/L is added into the residual bacterial liquid, and the bacterial liquid is placed on an oscillating table 220rpm and induced for 4 hours at 30 ℃. Taking 1ml of the induced bacterial liquid, centrifuging at 10000rpm for 10min, removing the supernatant, and preserving at-20 ℃.1ml PB was added to resuspend the sample as the solution to be tested. Taking 40 μl of solution to be detected, adding into 10 μl of 5×protein loading buffer, homogenizing in a metal bath, heating at 100deg.C for 5-10 min, cooling to room temperature, and instantaneously separating for 10s. 10 μl of the supernatant was subjected to 12% SDS-PAGE, and the results are shown in FIG. 2, and the SDS-PAGE result shows that: the recombinant engineering bacteria express his-GST-UreB protein, the molecular weight is 88kDa, and the molecular weight accords with the expectations.

S2, fermenting:

S21, starting strains: the fourth generation of pET 29-UreB/BL 21 seed bacteria is represented by 1: inoculating 600v/v ratio into first generation 2000 ml triangular flasks, each triangular flask containing LB medium 600 ml; placing the inoculated triangular flask in a constant-temperature shaking incubator at 36.0-38.0 ℃ for shaking culture for 4.0-8.0 hours at 200-240 rpm ℃ to obtain a first-generation production strain (this is the 5 th generation); after the culture is finished, taking a bacterial liquid sample for pure bacterial examination and microscopic examination, wherein the bacteria are gram-negative bacillus; the OD600 value of the bacterial liquid is more than 1.50.

S22, seed tank culture: inoculating the first-generation production bacteria into a seed tank according to the proportion of 1%, and filling LB culture medium into the seed tank with the filling amount of 30L; parameters of seed pot culture: temperature: 36.0-38.0 ℃; deep ventilation: 10.0 to 50.0L/min; rotational speed: 50-300 rpm parts; pH: 6.70-7.30; culturing time: 3.0 to 8.0 hours, which is a second generation production strain (this is the 6 th generation); when the cultivation is finished, taking a bacterial liquid sample for pure bacterial examination and microscopic examination, wherein the pure bacterial examination result is free of mixed bacteria, and the microscopic examination result is gram-negative bacillus; the OD600 value of the bacterial liquid is more than 1.50.

S23, culturing in a fermentation tank: inoculating the second-generation strain into a fermentation tank according to 10% of inoculation amount, and filling TB culture medium into the tank, wherein the filling amount is 270L; fermentation tank culture parameters: temperature: 36.0-38.0 ℃; deep ventilation: 50.0 to 350.0L/min; rotational speed: 100-500 rpm parts; pH: 6.70-7.30; culturing time: 3.0 to 7.0 hours; when the OD600 of the bacterial liquid is about 10.00, glycerol is started to be added, and the feeding speed is that: 0.015 L/min until the induction is finished; when the OD600 is about 20.00 (this is 7 th generation), the strain amplification is completed and the strain is ready to enter the induction stage.

S24, induction culture: when the OD600 of the bacterial liquid is about 20.00, the temperature of the fermentation tank is controlled to be 30.0 ℃, and the inducer solution subjected to sterilization and filtration is added according to the final concentration of 0.5 mmol to start induction; induction parameters: the temperature is 30.0deg.C (30.0deg.C); deep ventilation: 50.0 to 350.0L/min; rotational speed: 100-500 rpm parts; pH: 6.70-7.30; induction time 4.0 hours; at the beginning of induction, the concentrated culture medium is supplemented, and the feeding speed is that: 0.02 L/min until the induction is finished; and (3) after the culture is finished, taking a bacterial liquid sample for pure bacterial examination and microscopic examination, wherein the pure bacterial examination result is free of mixed bacteria, and the microscopic examination result is gram-negative bacillus.

S25, centrifugally collecting bacterial precipitate, transferring fermentation liquor into a bacterial liquor storage tank, centrifugally collecting bacterial with continuous flow (more than or equal to 12000 rpm), controlling the continuous flow liquid inlet speed to be 0.8-2.0L/min, and controlling the OD600 value of the centrifugal supernatant to be not more than 1.00; after centrifugation, the pellet (thallus) was collected; storing in the environment of-20.0 to-30.0 ℃.

S3, purification

S31, re-suspending the strain;

50mM Tris+0.15M NaCl+0.5% Tween80 in solution A, pH 8.0

And (3) adding the thallus and the solution A into the solution A according to the mass ratio of 1:10 for dilution and resuspension until the thallus is uniformly resuspended in the solution A.

S32, crushing thalli, centrifugally filtering and collecting supernatant;

the resuspended cells were subjected to shear homogenization: homogenizing at 2-8 ℃ for 3 times, wherein the temperature is 700-900 Bar;

Centrifuging the homogenized liquid under a centrifugal force of 12000g for 30min; collecting supernatant, clarifying and filtering with 0.6-0.8 μm deep layer filter plate, clarifying and filtering with 0.65 μm filter core, and purifying with column.

S33, ni-FF1 chromatography:

And (3) solution A: 50mM Tris+ 0.15M NaCl+0.5%Tween-80, pH 8.0

And (2) liquid B: 50mM Tris+0.15M NaCl+0.5M imidazole+0.5% Tween-80, pH 8.0

Balancing 2-3CV by adopting A liquid; loading a sample; the solution A is balanced for 3-5CV;7.5% of B liquid washing impurities for 2-5CV; samples were collected in a 30% B2-5 CV elution step.

S34, G25 liquid exchange:

And C, liquid: 50mM Tris+0.15M NaCl, pH 8.0

And C, liquid balancing, sample loading and C liquid eluting are adopted to collect samples, and the liquid exchange amount is less than or equal to 30% of the volume of the column bed each time.

S35, enzyme cutting: adding the ppase into the protein and the ppase according to the ratio of v/v=20:1, slightly stirring and mixing uniformly, and standing at 2-8 ℃ for overnight digestion; filtering, sampling and detecting.

S36, ni-FF2 chromatography:

And C, liquid: 50mM Tris+0.15M NaCl, pH 8.0

And D, liquid: 50mM Tris+0.15M NaCl+500mM imidazole, pH8.0

Balancing 2-3CV by adopting C liquid; loading a sample; the solution C is balanced for 2-5CV;4%D eluting with 20mM imidazole, 3-5CV; washing: 50% D solution, 250mM imidazole, 1-3CV.

S37, Q HP chromatography:

e, liquid: 20mM Tris+1mM DTT, pH 7.5

And F, liquid: 20mM Tris+0.5M L-Arg+1mM DTT, pH 7.5

Balance: the balance of the solution F is 1-2CV, and the balance of the solution E is 1-5CV; loading (sample diluted to conductance less than 8ms/cm using Buffer M); re-balancing 2-3CV; eluting with 0-100% F solution at 10CV, collecting eluting peak, sampling, and detecting.

S8, ultrafiltration liquid exchange, sterilization and filtration

G, liquid: 20mM PB+5 mM L-Cys Hcl+5% mannitol, pH 8.0

And (3) carrying out ultrafiltration concentration treatment on the three-step chromatographic sample liquid by using a 30KD membrane package. Concentrating to 1/5-1 times of the original volume. And adding G liquid in an equal volume, and continuously washing and filtering. Washing and filtering for 5-10 volumes, and concentrating the sample after dialysis to 1/2-2 times of the volume of the three-step chromatographic sample liquid. Under aseptic conditions, filtration was performed using a 0.22+0.45 μm filter. Split charging, sampling and detecting.

Example 2

(1) Experimental grouping and immunization procedure female BALB/c mice were randomly divided into 3 groups, 5 normal saline (blank) groups, the vaccine groups were intragastrically vaccinated with recombinant helicobacter pylori vaccine (e.coli), each mice was given the following prescribed vaccine (volumes all adjusted to 400 μl), and specifically, as shown in the following table, the challenge control group was immunized with 400 μl of normal saline. Gastric lavage immunity refers to standard operation procedure of pharmacodynamics experiments of oral recombinant helicobacter pylori vaccine (Escherichia coli), and is fed off in advance one day and water is cut off in the morning the next day.

Group of

Antigens

Immunization pathway

Immunization dose (μg/dose)

Immune volume (ml/only)

Immunization program (Tian)

Animal count (only)

1

Normal saline (blank control)

Oral administration

/

0.4

3 Times per week for 3 weeks

10

2

Normal saline (toxicity attack control)

Oral administration

/

0.4

3 Times per week for 3 weeks

10

3

Ureb

Oral administration

500

0.4

3 Times per week for 3 weeks

10

4

UreB+CpG

Oral administration

500+50

0.4

3 Times per week for 3 weeks

10

(2) Resuscitating and rejuvenating Hp strain

Resuscitating and rejuvenating Hp strain is started three days before virus attack, and the concentration of the bacterial liquid is adjusted to 1X 10 ⁷ CFU/ml.

(3) Attack toxin

And (3) after the fourth immunization, the drug is detoxified 10 days later, 0.4 ml/mouse is infused into the stomach, stomach tissues are killed and taken out two weeks after the drug is detoxified, the intragastric positioning condition (qPCR detection of the implantation amount) of the Hp mouse is detected, and the protection rate is calculated.

Immune protection test results

Group of	Antigens	Animal count (only)	Number of uninfected animals	Protection ratio (%)
					1	Normal saline (blank control)	10	10	-
2	Normal saline (toxicity attack control)	10	0	-
					3	Ureb	10	4	40%
4	UreB+CpG	10	6	60%

Example 3

Group of

Antigens

Immunization pathway

Immunization dose (μg/dose)

Immune volume (ml/only)

Immunization program (Tian)

Animal count (only)

1

Normal saline (blank control)

Oral administration

/

0.4

3 Times per week for 3 weeks

10

2

Ureb

Oral administration

500

0.4

3 Times per week for 3 weeks

10

3

UreB+CpG

Oral administration

500+50

0.4

3 Times per week for 3 weeks

10

Immunization

Female BALB/c mice were purchased for immunization, and the immunization was discontinued one day in advance and water was discontinued the next morning.

Pretreatment of immunization: and (3) immediately pouring the gastric acid neutralization solution (completing the whole immune process within 30 min), and continuing to eat for 2 hours to recover the food and water of the mice.

Sampling

After 14d of final immunization, tail blood was collected from each group, igG was detected, and stomach and intestine were taken for sIgA.

Sample detection

ELISA detection of serum IgG and intestinal tissue supernatant sIgA, determination of each group of coated UreB/HpaA [ UreB (5. Mu.g/ml) and HpaA (5. Mu.g/ml) ] based on the immune antigen

Serum was graded for eight, 1:50, 1:200, 1:800, 1:3200, 1:12800, 1:51200, 1:204800, and 1:819200, intestinal tissue supernatants were graded for eight, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280, gastric tissue supernatants were graded for eight, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280, vaginal washes were graded for 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 640.

ELIZA detection method

1) Experimental instrument

Pure water instrument (model ZYMICRO-II-20T Sichuan Zhuo water treatment equipment Co., ltd.), enzyme-labeled plate (model), enzyme-labeled instrument (model A51119600 Thermo FISHER SCIENTIFIC), micro plate washer (model 1575 Bio-Rad Laboratories), electric heating constant temperature incubator (model DHP-9052 Shanghai-Heng science instruments Co., ltd.), pipettor (model 10uL, 100uL, 200uL, 300uL, 1000uL, 5mL,10mLeppendorf Co., ltd.)

Experimental reagent

Purified water (resistivity no less than 18.25mΩ·cm), sodium carbonate (Na 2CO 3), sodium bicarbonate (NaHCO 3), disodium hydrogen phosphate 12 (Na 2hpo4·12h2o), sodium dihydrogen phosphate 2 (nah2po4·2h2o), sucrose, casein, BSA, proclin 300, aminopyrine, calf blood, polysorbate 20 (Tween 20), sodium chloride (NaCl), potassium chloride (KCl), potassium dihydrogen phosphate (KH 2PO 4), sodium merosal, TMB color development, sulfuric acid, HRP-labeled goat anti-mouse IgG secondary antibodies.

3) Experimental reagent preparation

Coating liquid: weighing Na2CO3 14.345g,NaHCO3 30.68g on an electronic balance, adding 500mL of distilled water, dissolving and mixing uniformly, and then fixing the volume to 1000mL, and marking: 10xCBS, and the coating is diluted by 10 times to 1xCBS with UP water.

Antibody dilution (enzyme dilution): on an electronic balance, 0.592g of Na2 HPO4. 12H2O 5.80g,NaH2PO4.2H2O, 5.00g of casein and 0.50g of aminopyrine are weighed, 500mL of distilled water is added, the materials are dissolved and mixed uniformly, 0.5mL of Tween20 is added by a pipette, the volume is fixed to 1000mL, and the calf serum 1000mL,Proclin 300 1mL is added, and the materials are mixed uniformly.

Washing liquid: dissolving 23.48 g/bag PBS in 2000 mL pure water, adding 1mL Tween 20, mixing well, sealing liquid: on an electronic balance, 0.59g of Na2 HPO4. 12H2O 5.80g,NaH2PO4.2H2O, 100.00g of sucrose, 1.00g of casein and 10.00g of BSA are weighed, 500mL of distilled water is added, dissolved and mixed uniformly, the volume is fixed to 1000mL, and 300 1mL of Proclin is added and mixed uniformly.

Stop solution (2 mol/L sulfuric acid): 443.9mL of ultrapure water was weighed into the reagent bottle, and then 56.1mL of concentrated sulfuric acid was slowly added by a pipette and thoroughly mixed.

Experimental method

(1) Coating: diluting the antigen to the required concentration (Ureb ug/mL coating concentration, hpaA coating concentration 5ug/mL, SS1 holoprotein 50 ug/mL), coating the ELISA plate with 100 mu L/hole, shaking and spreading thoroughly, and standing overnight at 4deg.C in a refrigerator or at 37deg.C 2 h.

(2) Closing: the plate was washed 3 times with washing liquid, 300. Mu.L each time, shaken 30 s, and pipetted 2.5. 2.5 s. Blocking solution 200. Mu.L/well blocking ELISA strips were placed in a refrigerator at 4℃overnight or at 37℃2 h.

(3) Adding primary antibody: the plate was washed 3 times with washing liquid, 300. Mu.L each time, shaken 30 s, and pipetted 2.5. 2.5 s. The serum of animal immunized by vaccine is diluted to 1:12800 by antibody diluent, and the serum obtained by animal immunized by normal saline is used as negative control, diluted according to the lowest dilution of serum of corresponding experimental group, shaking and shaking, and incubating for 2h at 37 ℃.

(4) Adding a secondary antibody: the plate was washed 3 times with washing liquid, 300. Mu.L each time, shaken 30 s, and pipetted 2.5. 2.5 s. HRP-labeled secondary anti-IgG was diluted 1:10000 times with antibody dilution, added at 100. Mu.L/well, shaken well and incubated for 1h at 37 ℃.

(5) Color development: the plate was washed 5 times with washing solution, 300. Mu.L each time, shaken 30 s, and pipetted 2.5. 2.5 s. 100. mu.L/well of TMB developing solution was added and developed 15 min in a dark place at 37℃in an incubator.

(6) Terminating the reaction: the reaction was terminated by adding 2mol/L H2SO4 to 50. Mu.L/well at the end of the development. The OD value of each well at 450 nm was measured using a microplate reader.

(7) The statistical method comprises the following steps: a sample/A negative >2.1 was used as positive standard.

Experimental results

1. Serum IgG, as shown in figure 3.

2. Diluted supernatant 1 in intestinal tract: 20 positive calculation of the conversion rate of yang

Group of	Antigens	Animal count (only)	Yang Zhuaishu A	Rate of positive rotation
					1	Normal saline (blank control)	10	0	0
2	Ureb	10	2	20%
					3	UreB+CpG	10	5	50%

Finally, it should be noted that:

The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. An oral helicobacter pylori vaccine recombinant protein antigen, which is characterized in that the antigen is UreB, and the amino acid sequence is shown in SEQ ID NO:1, the amino acid sequence of the fusion protein his-GST-UreB is shown as SEQ ID NO:2, the encoding nucleotide sequence of the fusion protein his-GST-UreB is shown as SEQ ID NO. 3.

2. The method for preparing recombinant protein antigen of oral helicobacter pylori vaccine according to claim 1, characterized by comprising the steps of:

S1, constructing a plasmid, and expressing protein: connecting a his-GST-UreB gene with a nucleotide sequence shown as SEQ ID NO. 3 into an expression vector, and transferring the constructed expression vector into recombinant engineering bacteria for induced expression;

s2, fermenting;

S3, purifying;

s32, crushing thalli, centrifugally filtering and collecting supernatant;

s38, ultrafiltration liquid exchange, sterilization and filtration are carried out to obtain UreB stock solution;

The solution A is 50mM Tris+0.15M NaCl+0.5%Tween-80, the pH is 8.0, the solution B is 50mM Tris+0.15M NaCl+0.5M imidazole+0.5% Tween-80, and the pH is 8.0; the solution C is 50mM Tris+0.15M NaCl and has the pH value of 8.0; the solution D is 50mM Tris+0.15M NaCl+500mM imidazole, and the pH value is 8.0; the E solution in S37 is 20mM Tris+1mM DTT,pH 7.5,F solution 20mM Tris+0.5M L-Arg+1mM DTT.

3. The method for preparing recombinant protein antigen of oral helicobacter pylori vaccine according to claim 2, characterized in that the expression vector in S1 is pET-29b (+), and the recombinant engineering bacterium is pET-UreB/BL21.

4. The method for preparing recombinant protein antigen of oral helicobacter pylori vaccine according to claim 2, characterized in that said S2 comprises the following substeps:

s25, centrifugally collecting bacterial precipitate.

5. The method for preparing recombinant protein antigen of oral helicobacter pylori vaccine according to claim 4, wherein the bacterial liquid sample is taken after the S21, S22 and S24 are finished and subjected to pure bacterial examination and microscopic examination, the bacteria are gram negative bacilli, and the bacterial liquid OD ₆₀₀ values after the S21, S22, S23 and S25 are finished are respectively more than 1.50, 20.00 and no more than 1.00; the S23 bacterial liquid OD ₆₀₀ starts to supplement glycerol at 0.015L/min at 10.00 until the induction is finished; the S24 induction was started by adding 0.02L/min of a medium containing 24.0 g/L of yeast extract and 12.0 g/L of tryptone.

6. The method for preparing recombinant protein antigen of oral helicobacter pylori vaccine according to claim 2, wherein the S32 is specifically: the resuspended cells were subjected to shear homogenization: homogenizing at 2-8 ℃ for 3 times, wherein the temperature is 700-900 Bar; centrifuging the homogenized liquid under a centrifugal force of 12000g for 30min; collecting supernatant, clarifying and filtering with 0.6-0.8 μm deep layer filter plate, and clarifying and filtering with 0.65 μm filter core.

7. The method for preparing recombinant protein antigen of oral helicobacter pylori vaccine according to claim 2, characterized in that the balancing in S33 is 2-3CV; loading a sample; re-balancing 3-5CV; washing: 7.5% B solution, 2-5CV; eluting: 30% B solution, 2-5CV.

8. The method for preparing recombinant protein antigen of oral helicobacter pylori vaccine according to claim 2, characterized in that the balancing is 2-3CV in S36; loading a sample; re-balancing 2-5CV; elution conditions: 4%D, 20mM imidazole, 3-5CV; washing: 50% D solution, 250mM imidazole, 1-3CV.

9. The method for preparing recombinant protein antigen of oral helicobacter pylori vaccine according to claim 2, wherein the ultrafiltration liquid exchange in S38 is three-step chromatographic sample liquid which is processed by ultrafiltration and concentration by a 30KD membrane package, the concentrated sample liquid is concentrated to 1/5-1 times of the original volume, the G liquid is added in equal volume, the continuous washing and filtering are carried out for 5-10 volumes, and the concentrated sample is dialyzed to 1/2-2 times of the volume of the three-step chromatographic sample liquid; the G solution is 20mM PB+5 mM L-Cys Hcl+5% mannitol, and the pH value is 8.0.

10. Use of an oral helicobacter pylori vaccine recombinant protein antigen according to claim 1 in the manufacture of a medicament for the prevention or treatment of helicobacter pylori infection.