CN115819625B

CN115819625B - Escherichia coli tetravalent antigen fusion polypeptide

Info

Publication number: CN115819625B
Application number: CN202211628639.5A
Authority: CN
Inventors: 张喜悦; 王君玮; 曲志娜; 王琳; 刘娜; 黄秀梅; 宋时萍
Original assignee: CHINA ANIMAL HEALTH AND EPIDEMIOLOGY CENTER
Current assignee: CHINA ANIMAL HEALTH AND EPIDEMIOLOGY CENTER
Priority date: 2022-12-18
Filing date: 2022-12-18
Publication date: 2024-02-13
Anticipated expiration: 2042-12-18
Also published as: CN115819625A

Abstract

The invention provides an escherichia coli tetravalent antigen fusion polypeptide, wherein the amino acid sequence of the antigen fusion polypeptide is SEQ ID NO. 1. The invention also provides lactococcus lactis for recombinant expression of the fusion polypeptide. The recombinant strain of the invention is used as vaccine for immunizing animals, and the content of the secretory IgA antibody, the content of the K88 antibody, the content of the K99 antibody and the content of the 987P, F antibody are obviously increased compared with the content of the control group. The fungus material prepared by the invention can produce a protective effect on most of pilus type diarrhea-causing escherichia coli causing diarrhea of pigs.

Description

Escherichia coli tetravalent antigen fusion polypeptide

Technical Field

The invention belongs to the technical field of antigen protein preparation, and particularly relates to an escherichia coli tetravalent antigen fusion polypeptide.

Background

The multi-epitope vaccine (multiepitope vaccine) is also called cocktail vaccine, and is a subunit vaccine designed based on the amino acid sequence of the target antigen epitope. The multi-epitope vaccine can be divided into a linear tandem multi-epitope vaccine, a multivalent antigen peptide epitope vaccine, a virus particle-like vaccine and the like. According to the different types of antigen epitope, the antigen epitope can be classified into B cell epitope vaccine, T cell epitope vaccine, mixed epitope vaccine and the like.

Colic diarrhea is an infectious disease with extremely high morbidity and mortality caused by enterotoxigenic escherichia coli, and causes serious economic loss to the pig industry. The piglets can have symptoms of diarrhea, dehydration, slow growth and the like, and even die suddenly. Meanwhile, the immunity of piglets is low, and other bacteria, viruses and the like are easy to be infected in a combined way, so that the normal development of the live pigs is affected. Colibacillosis has very obvious age characteristics, and can be classified into 3 kinds of piglet yellow dysentery, piglet white dysentery and piglet edema disease according to the difference of the age of the onset days. Yellow scour of piglets occurs within 7 days of age and has the characteristics of high morbidity and high mortality; white diarrhea of piglets occurs at 10-20 days old, and has the characteristics of high morbidity and low mortality; the piglet edema disease occurs 2 weeks after weaning, and has the characteristics of low morbidity and high mortality. The disease is characterized by enteritis and enterotoxemia, has no obvious seasonality, and is frequent in cold winter and hot and humid summer. The morbidity and mortality of the disease are affected by various factors such as the immune status of the pig flock, climate change, feeding management level, growth environment and the like. In order to ensure normal growth and slaughtering of live pigs, many farms and free-range farmers regularly use antibiotic drugs in the process of live pig cultivation

The composition can prevent and treat diseases, but the antibiotic medicine can be remained in the body of a pig after long-term administration or excessive use, so that the quality of pork products is affected, and soil and ecological environment can be destroyed due to the fact that antibiotics are contained in excretions of feces and urine. Meanwhile, the long-term use of a large number of antibiotics causes the enhancement of pathogenic bacteria resistance. Coli of porcine origin has been resistant to 19 antibiotics, the efficacy of which has been increasingly questioned.

The microecological preparation has no side effect, no residue and no resistance, is an ideal antibiotic substitute, and has wide application prospect. In recent years, the development of vaccines using lactic acid bacteria as a carrier has been attracting more and more attention. The lactobacillus oral vaccine can be directly taken orally without injection administration, so that the risk of damage infection caused by injection is avoided; can imitate natural infection path, induce mucosa immune response, and activate immune system; has immunological adjuvant effect, and the surface of lactobacillus can improve immunity and strengthen vaccine effect.

The basic principle of vaccine immunization is to use biological agents which can provoke immune response reaction, which is not directed against the whole exogenous substance but only against an epitope, usually a polypeptide, without causing injury, so that the organism obtains corresponding immunity. The epitope vaccine is a new vaccine developed in recent years, and is an epitope expressed in vitro or synthesized artificially by using genetic engineering means, and is used as a vaccine. Epitopes, also known as antigenic determinants, are chemical groups in antigenic molecules that determine the specificity of antigens, which are the basic units capable of specifically binding to the T cell antigen receptor TCR or the B cell antigen receptor BCR, ultimately eliciting an immune response in the body, forming an immune response against pathogenic microorganisms. According to different epitopes, epitope vaccines are classified into B epitope vaccines, T epitope vaccines and multi-epitope vaccines with both epitopes. The multi-epitope vaccine can induce B lymphocyte to produce antibody and activate cytotoxic lymphocyte to produce cell immune response, and has better application prospect.

Disclosure of Invention

The invention aims to provide an escherichia coli tetravalent antigen fusion polypeptide, which is prepared by fusing epitope peptides selected from escherichia coli K88, K99, 987P and F18 antigen genes, and can efficiently express the epitopes of the four genes in lactococcus lactis, so that the recombinant lactococcus lactis can be effectively and recombinantly expressed.

The invention provides a tetravalent antigen fusion polypeptide of escherichia coli, which comprises the following components:

1) A polypeptide with an amino acid sequence of SEQ ID NO. 1;

2) A polypeptide derived from 1) by substitution, deletion, addition of one or several amino groups on the sequence of 1);

the invention also provides a gene which codes the fusion polypeptide, and a nucleotide sequence of the gene is SEQ ID NO. 2;

in a further aspect, the present invention provides a recombinant expression vector into which a gene fragment encoding the fusion polypeptide is inserted;

the invention also provides a recombinant strain, which is used for recombinant expression of the fusion polypeptide;

the recombinant strain is recombinant lactococcus lactis.

In another aspect, the invention also provides an application of the fusion polypeptide or the recombinant strain in preparing vaccines.

In a further aspect, the invention provides a vaccine, wherein the antigen of the vaccine is a fusion polypeptide or a recombinant strain.

The recombinant strain of the invention is used as vaccine for immunizing animals, and the content of the secretory IgA antibody, the content of the K88 antibody, the content of the K99 antibody and the content of the 987P, F antibody are obviously increased compared with the content of the control group. The fungus material prepared by the invention can produce protective effect on the diarrhea-causing escherichia coli of most pilus types (K88, K99, 987 and P, F) causing diarrhea of pigs.

Drawings

FIG. 1 is a three-dimensional model diagram of a tetravalent multi-epitope fusion protein of Escherichia coli,

fig. 2: igA antibody detection results for immunized mice, wherein IgA antibody detection results for immunized mice are: p is less than 0.01, and the difference is extremely remarkable; in the graph, the OD value of IgA antibody detection of the experimental group of pNZ8149-8991/NZ3900 strain is obviously reduced compared with that of the control group of pNZ8149/NZ3900 lactobacillus and the blank control group, and the fact that the IgA antibody of mice in the experimental group is obviously increased and the difference is extremely obvious is proved;

fig. 3: k88, K99, 987P, F antibody detection results for immunized mice, wherein: p is less than 0.01, and the difference is extremely remarkable; in the figure, the OD values detected by the K88, K99 and 987P, F antibodies of the experimental group of the pNZ8149-8991/NZ3900 strain are remarkably increased compared with those of the control group of the pNZ8149/NZ3900 lactobacillus and the blank control group, and the difference is extremely remarkable.

Detailed Description

The adhesin is an important virulence factor of pathogenic escherichia coli, and can be adhered and planted in intestinal epithelial cells. The most important adhesins of E.coli are pili, including K88, K99, 987P and F18 pili. These pili are the most important pig coliform pili, which, once colonized the small intestine of piglets, produce large amounts of enterotoxins causing diarrhea. Wherein the amino acid sequences of the K88, K99, 987P and F18 genes are as follows:

k88ac Gene:

MKKTLIALAIAASAASGMAHAWMTGDFNGSVDIGGSITADDYRQKWEWKVVTGLNGFG

NVLNDLTNGGTKLTITVTGNKPLLLGRTKEAFATPVTGGVDGIPHIGFSEYEGGCVVVRKP

DGQTNKKGLAYFVLPMKNAEGTKVVSVKVNASYAGVLGRGGVTSADGELLSLFADGLSSIF

YGGLPRGSELSAGSAAAARTKLFGSLSRDDILGQIQRVNANNTSLVDVAGSYRENMQYTDGTVVSAAYALGIANGQTNEATFNQAVTTSTQWSAPLNLAITYY；

k99 gene:

NTGTINFNGKITSATCTIDPEVNGNRTSTIDLGQAAISGHGTVVDFKLKPAPGSNDC LAKTNARIDWSGSMNSLGFNNTASGNTAAKGYHMTLRATNVGNGSGGANINTSFTTAEYTH TSAIQSFNYSAQLKKDDRAPSNGGYKAGVFTTSASFLVTYM；

987P gene:

NTGTINFNGKITSATCTIDPEVNGNRTSTIDLGQAAISGHGTVVDFKLKPAPGSNDCL AKTNARIDWSGSMNSLGFNNTASGNTAAKGYHMTLRATNVGNGSGGANINTSFTTAEYTHT SAIQSFNYSAQLKKDDRAPSNGGYKAGVFTTSASFLVTYM；

f18 gene:

QQGDVKFFGSVSATTCNLTPQISGTVGDTIQLGTVTPNGTGSEIPFALKASSTAGGC ASLSNKTADITWSGQLTEKGFANQGGVANDSYVALKTVNGKTQAAQEVKASNSTVNFDASK ATTEGFKFTAQLKGGQTPGDFQGAAAYAVTYK。

however, if the amino acid sequences of the K88, K99, 987P and F18 genes are directly coupled, the expression efficiency of the coupled protein in lactococcus lactis is very low. The invention carries out molecular sequence analysis on cell epitopes of escherichia coli K88, K99, 987P and F18, couples selected antigen epitope polypeptides to form fusion polypeptides, clones the encoded nucleic acid fragments into escherichia coli-lactobacillus shuttle plasmid pNZ8149, and electrically converts the encoded nucleic acid fragments into lactococcus lactis NZ3900 to prepare the recombinant strain. The recombinant strain immunized mice are used for preparing clinical candidate vaccine of escherichia coli.

The present invention will be described in detail with reference to specific embodiments and drawings.

Example 1: construction of screening fusion proteins

1. Analysis of E.coli epitopes

B cell epitope, th cell epitope and CTL cell epitope analysis were performed on E.coli K88, K99, 987P and F18 genes, respectively.

(1) Analysis and screening of B cell antigen epitope polypeptides

The B cell epitopes of the K88ac genes of the E.coli K88, K99, 987P and F18 genes were predicted using IEDB and ABCpred on-line tools. The fragment with the overlapping predicted results is used as a target fragment, preferably a sequence with a longer fragment, and adjacent sequences are combined. Finally, 12 fragments are selected as E.coli B cell epitope amino acid fragments, and the related information is shown in Table 1.

Table 1: finally determined E.coli B cell antigen epitope polypeptide information table

(2) Th cell epitope prediction

The Th cell epitopes of K88, K99, 987P and 18F proteins were predicted, and the Th cell epitope peptide of K88 was determined to have an amino acid sequence of AYFVLPMKNAEGTK (designated K88-Th-CTL-129-142) and the amino acid sequence of VVDFKLKPAPGSNDCL (designated K99-Th-43-58). Whereas the Th cell epitope peptide of 987P coincides with 987P-B-36-88, the Th cell epitope peptide of F18 protein coincides with F18-B-95-144.

(3) Prediction of CTL cell epitopes

CTL cell epitopes of the K88, K99, 987P and 18F proteins were predicted. Wherein there are no dominant epitopes in the IEDB database; in NetMHC-4.0, molecule binding peptides HLA-A2, HLA-A 0201, HLA-A 0202, HLA-A 0203 and HLA-A 0205 are selected respectively, and a neural network and quantization matrix method (ANN+QM) is used, the threshold value is set to be 0.5, and the predicted molecule binding peptide overlapping sequences are synthesized to obtain 2 CTL epitopes (Table 2).

Table 2: CTL cell epitope prediction results table for K88, K99, 987P and 18F

Sequence number	Initiation site	Peptide fragment	Naming or remarks
				1	69	KLTITVTGNK	K88-CTL-69-78
2	120	AIQSFNYSAQLKK	987P-CTL-120-132

Example 2: construction and screening of tetravalent antigen fusion Polypeptides

Construction and screening of fusion polypeptides were performed from the identified multi-epitope peptide fragments of the K88, K99, 987P and F18 genes.

1. Construction of fusion polypeptide I

The 16 fragments are arranged in the order of 1 Th cell epitope, 3B cell epitope, 2B-CTL cell epitope, 1 Th cell epitope, 3B cell epitope, 1B-CTL cell epitope, 1B Th CTL cell epitope, 1B Th CTL cell epitope, and 1B CTL cell epitope.

The sequence is as follows: K88-Th-129-142, K88-B-48-69, K88-B-83-127, K88-B-249-280, K88-B-CTL-174-224, K99-B-CTL-94-153, K88-CTL-69-78, K99-Th-43-58, K99-B-22-88, 987P-B-15-30, F18-B-23-38, F18-B-CTL-49-65, 987P-CTL-120-132, F18-B-Th-95-144, F18-B-66-81, 987P-B-Th-TCL-36-88.

The N-terminal of the sequence is added with a general Th cell epitope (AKFVAAWTLKAAA), EAAAK is an N-terminal joint, PGPG is a Th cell epitope connecting linker, GKK is a B cell epitope connecting linker, and AAY is a CTL cell epitope linker. The antigenicity was predicted to be 0.9856 using VaxiJen on-line software.

2. Fusion polypeptide II

The 16 fragments were arranged in the order of 2 Th cell epitopes, 1B-Th cell epitope, 7B cell epitopes, 1B Th CTL cell epitope, 3B CTL cell epitopes, and 2 CTL cell epitopes, and the amino acid fragments of the cell epitopes were arranged.

The sequence is as follows:

K88-Th-129-142、K99-Th-43-58、F18-B-Th-95-144、K88-B-48-69、K88-B-83-127、K88-B-249-280、K99-B-22-88、987P-B-15-30、F18-B-23-38、F18-B-66-81、987P-B-Th-TCL-36-88、K88-B-CTL-174-224、K99-B-CTL-94-153、F18-B-CTL-49-65、K88-CTL-69-78、987P-CTL-120-132。

the N-terminal of the above sequence was added with a universal Th cell epitope (AKFVAAWTLKAAA), and the linker sequence was ligated to polypeptide I. The antigenicity was predicted to be 0.9885 using VaxiJen on-line software.

3. Fusion polypeptide III

The 16 fragments were arranged in the order of K88-K99 cell epitope (B cell epitope, B cell-CTL cell fusion epitope, CTL cell epitope, th cell-CTL cell fusion epitope), 987P-F18 cell epitope (B cell epitope, B cell-Th cell epitope, CTL cell epitope, B cell-CTL cell fusion epitope, B cell-Th cell-CTL cell fusion epitope).

The sequence is as follows: K88-B-48-69, K88-B-83-127, K88-B-249-280, K99-B-22-88, K88-B-CTL-174-224, K99-B-CTL-94-153, K88-CTL-69-78, K99-Th-43-58, K88-Th-CTL-129-142, 987P-B-15-30, F18-B-23-38, F18-B-66-81, F18-B-Th-95-144, 987P-CTL-120-132, F18-B-CTL-49-65, 987P-B-Th-TCL-36-88.

The N-terminal of the above sequence is added with a general Th cell epitope (AKFVAAWTLKAAA), EAAAK is N-terminal linker, and the linker sequence is connected with polypeptide I. The antigenicity was predicted to be 0.9822 using VaxiJen on-line software.

The 3 linked polypeptides were compared, with the highest antigenicity of linked polypeptide II and the lowest antigenicity of linked polypeptide III. Allergy prediction was performed on the linked polypeptide ii using AllerTOP v.2.0 online tool, and as a result the protein was not allergic. The protein is subjected to three-dimensional modeling by using an I-TASSER online tool, has good structural epitope exposure, is easy to combine with an antibody, and meets the epitope design requirement on the protein molecular conformation (figure 1); the figure shows that the protein domains have less interference with each other, the domains on the surface are rich, and the structural epitope has better exposure.

The amino acid sequence of the finally determined fusion polypeptide II is as follows (SEQ ID NO: 1):

AKFVAAWTLKAAAEAAAKAYFVLPMKNAEGTKPGPGVVDFKLKPAPGSNDCLPGPGV

NGKTQAAQEVKASNSTVNFDASKATTEGFKFTAQLKGGQTPGDFQGAAAPGPGWKVVTGLN

GFGNVLNDLTNGGTGKKLGRTKEAFATPVTGGVDGIPHIGFSEYEGGCVVVRKPDGQTNKK

GGKKALGIANGQTNEATFNQAVTTSTQWSAPLNLAIGKKVNGNRTSTIDLGQAAISGHGTV

VDFKLKPAPGSNDCLAKTNARIDWSGSMNSLGFNNTASGNTAAKGGKKTCTIDPEVNGNRT

STIGKKSGTVGDTIQLGTVTPNGKKDITWSGQLTEKGFANQGKKAISGHGTVVDFKLKPAP

GSNDCLAKTNARIDWSGSMNSLGFNNTASGNTAAKGAAYDGLSSIFYGGLPRGSELSAGSA

AAARTKLFGSLSRDDILGQIQRVNANNTSAAYRATNVGNGSGGANINTSFTTAEYTHTSAI

QSFNYSAQLKKDDRAPSNGGYKAGVFTTSASAAYKASSTAGGCASLSNKTAAAYKLTITVTGNKAAYAIQSFNYSAQLKK(SEQ ID NO：1)。

wherein, the italic part is Th general cell epitope site, and the underlined part is Linker.

The amino acid of the multi-epitope vaccine is optimized for the preference of the codon of the lactic acid bacteria, and the base sequence of the finally determined multi-epitope peptide fragment is as follows (SEQ ID NO: 2):

GCTAAATTTGTTGCTGCTTGGACATTAAAAGCTGCTGCTGAAGCTGCTGCTAAAGCTTATTTTGTTTTACCAATGAAAAATGCTGAAGGTACAAAACCAGGTCCAGGTGTTGTTGATTTTAAATTAAAACCAGCTCCAGGTTCAAATGATTGTTTACCAGGTCCAGGTGTTAATGGTAAAACACAAGCTGCTCAAGAAGTTAAAGCTTCAAATTCAACAGTTAATTTTGATGCTTCAAAAGCTACAACAGAAGGTTTTAAATTTACAGCTCAATTAAAAGGTGGTCAAACACCAGGTGATTTTCAAGGTGCTGCTGCTCCAGGTCCAGGTTGGAAAGTTGTTACAGGTTTAAATGGTTTTGGTAATGTTTTAAATGATTTAACAAATGGTGGTACAGGTAAAAAATTAGGTCGTACAAAAGAAGCTTTTGCTACACCAGTTACAGGTGGTGTTGATGGTATTCCACATATTGGTTTTTCAGAATATGAAGGTGGTTGTGTTGTTGTTCGTAAACCAGATGGTCAAACAAATAAAAAAGGTGGTAAA

AAAGCTTTAGGTATTGCTAATGGTCAAACAAATGAAGCTACATTTAATCAAGCTGTTACAA

CATCAACACAATGGTCAGCTCCATTAAATTTAGCTATTGGTAAAAAAGTTAATGGTAATCG

TACATCAACAATTGATTTAGGTCAAGCTGCTATTTCAGGTCATGGTACAGTTGTTGATTTT

AAATTAAAACCAGCTCCAGGTTCAAATGATTGTTTAGCTAAAACAAATGCTCGTATTGATT

GGTCAGGTTCAATGAATTCATTAGGTTTTAATAATACAGCTTCAGGTAATACAGCTGCTAA

AGGTGGTAAAAAAACATGTACAATTGATCCAGAAGTTAATGGTAATCGTACATCAACAATT

GGTAAAAAATCAGGTACAGTTGGTGATACAATTCAATTAGGTACAGTTACACCAAATGGTA

AAAAAGATATTACATGGTCAGGTCAATTAACAGAAAAAGGTTTTGCTAATCAAGGTAAAAA

AGCTATTTCAGGTCATGGTACAGTTGTTGATTTTAAATTAAAACCAGCTCCAGGTTCAAAT

GATTGTTTAGCTAAAACAAATGCTCGTATTGATTGGTCAGGTTCAATGAATTCATTAGGTT

TTAATAATACAGCTTCAGGTAATACAGCTGCTAAAGGTGCTGCTTATGATGGTTTATCATC

AATTTTTTATGGTGGTTTACCACGTGGTTCAGAATTATCAGCTGGTTCAGCTGCTGCTGCT

CGTACAAAATTATTTGGTTCATTATCACGTGATGATATTTTAGGTCAAATTCAACGTGTTA

ATGCTAATAATACATCAGCTGCTTATCGTGCTACAAATGTTGGTAATGGTTCAGGTGGTGC

TAATATTAATACATCATTTACAACAGCTGAATATACACATACATCAGCTATTCAATCATTT

AATTATTCAGCTCAATTAAAAAAAGATGATCGTGCTCCATCAAATGGTGGTTATAAAGCTG

GTGTTTTTACAACATCAGCTTCAGCTGCTTATAAAGCTTCATCAACAGCTGGTGGTTGTGC

TTCATTATCAAATAAAACAGCTGCTGCTTATAAATTAACAATTACAGTTACAGGTAATAAAGCTGCTTATGCTATTCAATCATTTAATTATTCAGCTCAATTAAAAAAATAA。

the above sequences were synthesized by Shanghai Biotechnology Co., ltd and cloned into the pET28a plasmid, designated pET28a-8991.

Example 3: construction of recombinant lactococcus lactis

1. Amplification of the target fragment.

The pET28a-XDT plasmid was used as a template, and XDT-F, XDT-R was used as a primer to amplify the signal peptide sequence (primer sequences are shown in Table 3). Plasmid was extracted from strain pET28a-8991 using a plasmid extraction kit, and the nucleic acid sequences of multivalent epitope peptides were amplified using 8991-F and 8991-R primers as templates (primer sequences are shown in Table 3). The CR reaction system and conditions were as follows: the PCR reaction system was 20. Mu.L, including 2×Taq Master Mix 10. Mu.L, primer F1. Mu.L, primer R1. Mu. L, vector 1. Mu. L, ddH ₂ O7. Mu.L. The reaction procedure: pre-denaturation at 95℃for 5min; denaturation at 95℃for 1min, annealing at 46℃for 1min, extension at 72℃for 90s for a total of 35 cycles; final extension at 72℃for 10min.

Table 3: PCR primer sequence table for amplifying target fragment

Extraction and cleavage of the pNZ8149 plasmid

Inoculating pNZ8149/NZ3900 strain (laboratory preservation) strain into GM17 culture medium, standing at 30deg.C for overnight culture, collecting 2mL bacterial solution, and using gram-positivePlasmid pNZ8149 was extracted using the sex-fungus plasmid miniprep kit (Solarbio). Using restriction enzymes SacI and NcoI, the system was 2. Mu.L NEB buffer, 8. Mu.L pNZ8149 plasmid, 1. Mu.L Sac1, 1. Mu.L Nco1, 8. Mu.L ddH ₂ 0. After heating in water bath at 37 ℃ for 3 hours, the enzyme-digested product is detected by agarose gel electrophoresis, and a gel recovery kit (Omega) is used for recovering and purifying the target band for carrier construction.

3. Construction of recombinant lactic acid bacteria strains

The amplified XDT sequence fragment and 8991 sequence fragment were recovered by a gel recovery kit, mixed with the double digested and purified pNZ8149 plasmid fragment, and added with 1.5. Mu.L of the seamless cloning PreMix Premix (Monad) 5. Mu. L, XDT fragment, 1.5. Mu.L of the 8991 fragment and 2. Mu.L of the recovered pNZ8149 plasmid, and subjected to water bath at 37℃for 25 minutes. Mixing 5 μl of the above connection product with 50 μl of NZ3900 competent cells, incubating on ice for 15min, transferring to a pre-cooled electric shock cup (1 mm) for 15min, electric shocking at 1250V, rapidly adding 950 μl of resuscitation medium, and allowing to act at 30deg.C for 1.5-2 hr. 100 mu L of the recombinant plasmid is inoculated on an ELIKER plate, cultured for 2 days at 30 ℃, yellow single colonies are picked up to 5mL of liquid culture medium, the culture is carried out at 30 ℃ for overnight, a positive bacteria plasmid extraction kit is used for extracting the recombinant plasmid pNZ8149-8991, and the sequence of the recombinant plasmid is correct through sequencing. The construction of pNZ8149-8991/NZ3900 strain was confirmed to be successful.

4. Stability test of recombinant strains

The recombinant strain pNZ8149-8991/NZ3900 is passaged in M17 (0.5% lactose) culture medium, inoculated in an Eliker screening culture medium after 20 generations, more than 98% of colonies are still positive colonies, and the plasmid is extracted and identified to be correct through sequencing. It was shown that more than 97% of the recombinant strain remained stable after 20 passages (see Table 4).

Table 4: positive colony ratio table (unit: 100%) after passage of parent and recombinant strain

Times (generation)	0	5	10	15	20
						pNZ8149/NZ3900	100	99	99	98	98
pNZ8149-8991/NZ3900	100	98	98	97	97

Taking no-load bacteria as control group and recombinant bacteria as test group, inoculating into M17 (0.5% lactose) culture medium, and measuring OD every half hour ₆₀₀ (see Table 5), the growth curves of the two are drawn and compared, and the growth curves of the recombinant bacteria and the parent bacteria are basically consistent; meanwhile, through t test, the growth curves of the two are not obviously different. It was confirmed that the growth performance of the recombinant bacteria was not affected compared with the parent bacteria.

Table 5: OD of parent and recombinant strains for different growth times ₆₀₀ Value table

5. Preparation of lactobacillus material for immunization

The identified lactobacillus pNZ8149-8991/NZ3900 is inoculated into 5mLGM17 liquid culture medium, and the lactobacillus is subjected to static culture at the constant temperature of 30 ℃ for overnight. The next day, 5mL of the activated bacterial liquid was inoculated into 200mLGM17 medium for further culture. Culturing to OD ₆₀₀ When the concentration is 0.3-0.4, adding Nisin solution with the final concentration of 10ng/mL, standing at 30 ℃ for induction for 6 hours, and mixing bacterial liquid with 2% sodium alginate solution (the mixing ratio is 1:2); adding starch with final concentration of 4%, fructose with final concentration of 4%, glucose with final concentration of 1%, and whole egg liquid with final concentration of 200mL/L, mixing completely, and dripping into CaCl with final concentration of 2% ₂ Gel beads are generated in the solution, the gel beads are washed for 3 times by pure water after filtration, and the gel beads are solidified and shaped for 30 to 60 minutes. Filtering, washing with pure water for 3 times, adding into 1% chitosan solution (the ratio of the chitosan solution to lactobacillus is 1:1), stirring, coating for 30-60 min, filtering, washing for 3 times, and air drying at room temperature.

6. Immunization of lactic acid bacteria material

Taking the dried fungus material, adding an M17 liquid culture medium, standing for 10min, homogenizing by a beating type homogenizer, and counting by a plate colony counting method (or turbidimetry method) after homogenizing. According to the counting result, the concentration of the original bacterial liquid is adjusted to ensure that the immunity concentration of the bacterial material is 5 multiplied by 10 ⁹ cfu/g, immunized mice were fed quantitatively.

18 BABL/c mice were randomly divided into 3 groups A-F, each group being 6. Wherein the first group was set as a blank, and no vaccine was immunized; the second group is set as lactobacillus strain control, and immune empty carrier pNZ8149/NZ3900 lactobacillus material is quantitatively fed; the third group is set as NZ8149-8991/NZ3900 lactobacillus strain test group, and immune NZ8149-8991/NZ3900 strain is quantitatively fed. The immunization procedure was: priming the mice at 1d, 2d, 3d, 4d, 5d, continuous immunization for 5 days; mice were immunized 2 times at 30d, 31d, 32d, 33d, 34d for 5 days; at 59d, 60d, 61d, 62d, 6The mice were boosted a third time for 3d and immunized for 5 consecutive days. The immune dose is as follows: each mouse was fed 5X 10 quantitatively ⁹ cfu/g bacteria material 2g. After 25 days of final immunization, all 18 mice were sacrificed and serum was collected; and shearing and retrieving the small intestine of the intestinal segment by 5cm, flushing intestinal mucosa by using 200 mu L of small intestine lavage liquid, and collecting intestinal juice.

Serum and small intestine lavage fluid samples are detected by referring to the mouse K88 antibody ELISA detection kit, the K99 antibody detection kit, the 987P antibody ELISA detection kit, the F18 antibody ELISA detection kit and the secretory IgA antibody ELISA detection kit instruction. The IgA antibody ELISA detection kit is a competitive ELISA kit, and through detection, the IgA antibody of the mice in the experimental group is found to be obviously reduced in OD value compared with the control group (see figure 2), and the IgA antibody of the mice in the experimental group is proved to be obviously increased; the K88 antibody, the K99 antibody, the 987P antibody and the F18 antibody of the experimental group were also significantly elevated compared to the control group (see fig. 3).

Meanwhile, the laboratory uses the amino acid sequence of the connecting polypeptide to reversely translate into nucleotide sequence, and simultaneously passes through the company to clone the upstream and downstream sequences, uses the lactococcus lactis to clone and express the connecting polypeptide, and immunizes mice, and as a result, the connecting polypeptide I and the connecting polypeptide III stimulate the K88 antibody produced by the mice, the titer of the K88 antibody is lower than that of the antibody produced by the connecting polypeptide II stimulated by the mice, and the antigenicity of the connecting polypeptide II is higher than that of the connecting polypeptide I and the connecting polypeptide III, and is consistent with the antigenicity result predicted by software. Since the diarrhea causing escherichia coli infects animals mainly through the digestive tract, severe damage to intestinal mucosa can be caused.

Claims

1. The escherichia coli tetravalent antigen fusion polypeptide is characterized in that the amino acid sequence of the fusion polypeptide is SEQ ID NO. 1.

2. A gene encoding the fusion polypeptide of claim 1.

3. The gene according to claim 2, wherein the nucleotide sequence of the gene is SEQ ID NO. 2.

4. A recombinant expression vector having inserted therein a gene segment encoding the fusion polypeptide of claim 1.

5. A recombinant strain, wherein the recombinant strain is transformed or transfected with the recombinant expression vector of claim 4.

6. The recombinant strain of claim 5, wherein the recombinant strain is a recombinant lactococcus lactis.

7. Use of the fusion polypeptide of claim 1 for the preparation of a vaccine for preventing diarrhea in piglets caused by enterotoxigenic escherichia coli.

8. Use of the recombinant strain of claim 5 in the preparation of a vaccine for preventing diarrhea in piglets caused by enterotoxigenic escherichia coli.

9. A vaccine for preventing diarrhea in piglets caused by enterotoxigenic escherichia coli, wherein the antigen of the vaccine comprises the fusion polypeptide of claim 1.

10. The vaccine of claim 9, wherein said antigen further comprises the recombinant strain of claim 5.