CN115725002B - Coli specific antigen fusion protein and recombinant lactococcus lactis thereof - Google Patents

Abstract

The invention provides an escherichia coli specific antigen fusion protein and recombinant lactococcus lactis thereof, and the fusion polypeptide provided by the invention has an amino acid sequence of SEQ ID NO:1, a step of; a specific nucleotide sequence of the coding gene is SEQ ID NO. 2. The fusion polypeptide provided by the invention can be used as an antigen to prepare a vaccine, and the prepared vaccine can provide high-efficiency immune protection after animals are immunized. Furthermore, the fusion polypeptide provided is not allergic.

Description

Coli specific antigen fusion protein and recombinant lactococcus lactis thereof

Technical Field

The invention belongs to the technical field of biological products for animals, and particularly relates to an escherichia coli specific antigen fusion protein and recombinant lactococcus lactis thereof.

Background

Diarrhea in piglets is one of the most common diseases in pig farms on a global scale, and brings great losses to the pig industry. Enterotoxigenic escherichia coli (ETEC) is a main pathogenic bacterium causing diarrhea of piglets, adhesins and enterotoxigenic substances are important pathogenic factors, and the ETEC is fixedly planted in small intestinal epithelial cells through the adhesins, so that enterotoxigenic substances are continuously generated in the proliferation process, a large amount of water and electrolyte are caused to enter the intestinal cavity, and diarrhea of the piglets is caused. At present, vaccine immunization is the most effective method for preventing ETEC, but ETEC has numerous serotypes, poor cross immune protection effect, complex pathogenic factors and easy mutation, so that the existing vaccine on the market can provide better protection on test animals, and the actual application effect is often poor. Therefore, the development of safe, efficient and broad-spectrum ETEC vaccine has important significance for pig industry.

Probiotics are active microorganisms, are the general term for active microorganisms which are planted in animal intestinal tracts and reproductive systems and can generate definite health effects so as to improve host micro-ecological balance and play a beneficial role. Probiotics in a broad sense cover a plurality of microorganisms of families and genera, and so far more than 90 species are classified into lactic acid bacteria, bacillus and yeasts, and among them, lactic acid bacteria are the most widely used. In recent years, with the development of live vector vaccines, lactic acid bacteria have been attracting more and more attention as a vector for expressing foreign proteins. The live carrier vaccine of lactic acid bacteria has high safety performance and good physiological activity, is a good adjuvant, can regulate the immune level of organisms and improve the immunogenicity of antigens, and is convenient to use by immunization in a nasal drip or oral mode.

Disclosure of Invention

The invention aims to provide an escherichia coli specific antigen fusion protein and recombinant lactococcus lactis thereof, wherein the antigen fusion protein is escherichia coli K88-K99 multivalent specific antigen fusion protein, and the recombinant lactococcus lactis is used for recombinant expression of the fusion protein.

The present invention first provides a fusion polypeptide comprising:

1) The amino acid sequence is SEQ ID NO:1;

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

the invention also provides a gene which codes for the fusion polypeptide;

one specific nucleotide sequence of the gene is SEQ ID NO. 2.

In a further aspect, the present invention provides a recombinant expression vector into which the nucleotide fragment of the above-described coding gene is inserted;

the recombinant expression vector is a pNZ8149 plasmid expression vector;

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

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 fusion polypeptide provided by the invention can be used as an antigen to prepare a vaccine, and the prepared vaccine can provide high-efficiency immune protection after animals are immunized. The fusion polypeptide provided has no allergy, does not cause allergy of immunized animals, and ensures the safety of the fusion polypeptide as a vaccine.

Drawings

Fig. 1: the three-dimensional model diagram of the multi-epitope fusion protein shows that the protein domains have less interference with each other, the domains on the surface are rich, and the structural epitope exposure is good;

fig. 2: a stability test result graph of the recombinant strain;

fig. 3: a graph of growth of the recombinant strain;

fig. 4: igA antibody detection results for immunized mice are shown in the following figures: 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 the pNZ8149-KT/NZ3900 strain is reduced by more than 1.5 compared with that of the control group of the pNZ8149/NZ3900 lactobacillus and the blank control group, and the IgA antibody of mice in the experimental group is proved to be increased by more than 10 times and the difference is very obvious;

fig. 5: graph of K88 antibody detection results for immunized mice, wherein: p is less than 0.01, and the difference is extremely remarkable; in the graph, the OD value of the K88 antibody detection of the pNZ8149-KT/NZ3900 strain experimental group is obviously increased and the difference is extremely obvious compared with that of a pNZ8149/NZ3900 lactobacillus control group and a blank control group;

fig. 6: graph of K99 antibody detection results for immunized mice, wherein: p is less than 0.01, and the difference is extremely remarkable; in the graph, the OD read value of the K99 antibody detection of the pNZ8149-KT/NZ3900 strain experimental group is remarkably increased compared with that of a pNZ8149/NZ3900 lactobacillus control group and a blank control group, and the difference between the experimental group and the other two control groups is extremely remarkable.

Detailed Description

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.

After molecular sequence analysis is carried out on cell epitopes of escherichia coli K88 and K99, selected antigen epitope polypeptides are coupled to form fusion proteins, encoding nucleic acid fragments of the fusion proteins are cloned into escherichia coli-lactobacillus shuttle plasmid pNZ8149, the encoding nucleic acid fragments are electrically transformed into lactococcus lactis NZ3900, bacterial materials are prepared, and mice are immunized and used for preparing escherichia coli clinical candidate vaccines.

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, such as K88 pili, K99 pili, which, once colonized the small intestine of piglets, produce large amounts of enterotoxins causing diarrhea. The K88 pili are classified into K88ab, K88ac and K88ad, wherein K88ac and K99 are main popular pili types in China, and the amino acid sequence information is as follows:

k88ac Gene:

MKKTLIALAIAASAASGMAHAWMTGDFNGSVDIGGSITADDYRQKWEWKVVTGLNGFGNVLNDLTNGGTKLTITVTGNKPLLLGRTKEAFATPVTGGVDGIPHIGFSEYEGGCVVVRKPDGQTNKKGLAYFVLPMKNAEGTKVVSVKVNASYAGVLGRGGVTSADGELLSLFADGLSSIFYGGLPRGSELSAGSAAAARTKLFGSLSRDDILGQIQRVNANNTSLVDVAGSYRENMQYTDGTVVSAAYALGIANGQTNEATFNQAVTTSTQWSAPLNLAITYY；

k99 gene:

NTGTINFNGKITSATCTIDPEVNGNRTSTIDLGQAAISGHGTVVDFKLKPAPGSN DCLAKTNARIDWSGSMNSLGFNNTASGNTAAKGYHMTLRATNVGNGSGGANINTS FTTAEYTHTSAIQSFNYSAQLKKDDRAPSNGGYKAGVFTTSASFLVTYM。

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

Example 1: construction of screening fusion proteins

1. Analysis and screening of E.coli K88ac B cell antigen epitope polypeptide

B cell epitopes of the E.coli K88ac gene were analyzed, wherein the IEDB predictions for K88ac are shown in Table 1 and the ABCpred predictions are shown in Table 2.

Table 1: IEDB predictive result table of K88ac B cell antigen epitope

Sequence number	Initiation site	Termination site	Peptide fragment	Length of
					1	23	31	MTGDFNGSV	9
2	38	46	TADDYRQKW	9
					3	48	69	WKVVTGLNGFGNVLNDLTNGGT	22
4	88	101	EAFATPVTGGVDGI	14
					5	105	127	GFSEYEGGCVVVRKPDGQTNKKG	23
6	160	167	GVTSADGE	8
					7	174	224	DGLSSIFYGGLPRGSELSAGSAAAARTKLFGSLSRDDILGQIQRVNANNTS	51
8	229	247	AGSYRENMQYTDGTVVSAA	19
					9	250	257	LGIANGQT	8
10	262	272	FNQAVTTSTQW	11

Table 2: ABCpred predictive outcome table for K88ac B cell epitope

Rating of	Initiation site	Peptide fragment	Scoring of
				1	144	VSVKVNASYAGVLGRGGV	0.88
2	184	LPRGSELSAGSAAAARTK	0.87
				2	165	DGELLSLFADGLSSIFYG	0.87
3	249	ALGIANGQTNEATFNQAV	0.86
				4	48	WKVVTGLNGFGNVLNDLT	0.85
5	96	GGVDGIPHIGFSEYEGGC	0.83
				6	58	GNVLNDLTNGGTKLTITV	0.82
6	125	KKGLAYFVLPMKNAEGTK	0.82
				7	83	LGRTKEAFATPVTGGVDG	0.80
8	263	NQAVTTSTQWSAPLNLAI	0.79

The target fragment is a fragment in which the IEDB predicted result and the ABCpred predicted result of the K88ac epitope polypeptide coincide, preferably a sequence in which the fragment is longer, and adjacent sequences are combined. Finally, 4 fragments are selected as K88ac epitope polypeptide fragments, and the related information is shown in table 3.

Table 3: finally determined K88ac B cell antigen epitope polypeptide information table

2. The B cell epitope of the E.coli K99 gene was analyzed, wherein the IEDB predicted results for the K99B cell epitope are shown in Table 4 and the K99 predicted results are shown in Table 5.

Table 4: k99 IEDB predictive result table of B cell antigen epitope polypeptide

Sequence number	Initiation site	Termination site	Peptide fragment	Length of
					1	20	29	PEVNGNRTST	10
2	36	88	AISGHGTVVDFKLKPAPGSNDCLAKTNARIDWSGSMNSLGFNNTASGNTAAKG	53
					3	96	114	TNVGNGSGGANINTSFTTA	19
4	118	126	HTSAIQSFN	9
					5	132	152	KKDDRAPSNGGYKAGVFTTSA	21

Table 5: k99 ABCpred prediction result table of B cell antigen epitope polypeptide

Rating of	Initiation site	Peptide fragment	Scoring of
				1	83	NTAAKGYHMTLRATNVGN	0.87
2	72	NSLGFNNTASGNTAAKGY	0.84
				2	55	NDCLAKTNARIDWSGSMN	0.84
2	113	TAEYTHTSAIQSFNYSAQ	0.84
				3	94	RATNVGNGSGGANINTSF	0.78
4	62	NARIDWSGSMNSLGFNNT	0.77
				4	22	VNGNRTSTIDLGQAAISG	0.77
5	136	RAPSNGGYKAGVFTTSAS	0.76

And taking a fragment, which is formed by overlapping the IEDB predicted result and the ABCpred predicted result of the K99B epitope polypeptide, as a target fragment, preferably a sequence with a longer fragment, and combining adjacent sequences. Finally, 2 fragments are selected as K99B epitope polypeptides, and related information is shown in Table 6.

Table 6: finally determined K99B cell antigen epitope polypeptide information table

3. Th cell epitope prediction

Th cell epitope of K88ac and K99 proteins is predicted, and the amino acid sequence of Th cell epitope peptide of K88ac is AYFVLPMKNAEGTK (named as K88-Th-CTL-129-142). K99 The amino acid sequence of the Th cell epitope peptide was VVDFKLKPAPGSNDCL (designated K99-Th-43-58).

4. Prediction of CTL cell epitopes

CTL epitopes of K88, K99 proteins were predicted. Wherein there are no dominant epitopes in the IEDB database; in NetMHC-4.0, molecular 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 a quantization matrix method (ANN+QM) are used, the threshold value is set to be 0.5, and the predicted molecular binding peptide overlapping sequences are synthesized to obtain 1 CTL epitope (table).

Table 7: cT cell epitope predictive outcome table for K88

Sequence number	Initiation site	Peptide fragment	Naming or remarks
				1	69	KLTITVTGNK	K88-CTL-69-78
2	126	GLAYFVLPMK	Coincident with K88 Th cells
				3	196	AARTKLFGSL	Coincidence with K88-B cell epitope

Table 8: CTL cell epitope prediction result table of K99

Sequence number	Initiation site	Peptide fragment	Naming or remarks
				1	122	QSFNYSAQLK	Coincidence with K99-B cell epitope
2	106	INTSFTTAEY	Coincidence with K99-B cell epitope
				3	117	HTSAIQSFNY	Coincidence with K99-B cell epitope
4	148	TTSASFLVTY	Coincidence with K99-B cell epitope

(III) ligation of polyepitope peptide fragments

1. Ligation polypeptide I

The 9 fragments are arranged according to the sequence of Th cell epitope, B cell epitope, th cell-CTL cell fusion epitope, B cell-CTL cell fusion epitope and CTL cell epitope.

The connection sequence is as follows:

K99-Th-43-58、K88-B-48-69、K88-B-83-127、K88-B-249-280、K99-B-22-88、K88-Th-CTL-129-142、K88-B-CTL-174-224、K99-B-CTL-94-153、K88-CTL-69-78。

adding a general Th cell epitope (AKFVAAWTLKAAA) on the N end of the sequence, wherein EAAAK is an N-end 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 0.8987 using VaxiJen on-line software.

2. Ligation polypeptide II

The 9 fragments are arranged according to the sequence of B cell epitope, B cell-CTL cell fusion epitope, CTL cell epitope, th cell epitope and 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.

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, and its antigenicity was predicted to be 0.9068 using VaxiJen on-line software.

3. Ligation polypeptide III

The 9 fragments, namely, the CTL cell epitope, the Th cell-CTL cell fusion epitope, the B cell-CTL cell fusion epitope, the Th cell epitope and the B cell epitope are arranged in sequence, and the amino acid fragments of the cell epitopes are arranged.

The sequence is as follows: K88-CTL-69-78, K88-Th-CTL-129-142, K88-B-CTL-174-224, K99-B-CTL-94-153, K99-Th-43-58, K88-B-48-69, K88-B-83-127, K88-B-249-280, K99-B-22-88.

Adding a universal Th cell epitope (AKFVAAWTLKAAA) to the N-terminal of the sequence, wherein EAAAK is an N-terminal connector, and the connector sequence is connected with the polypeptide I; the antigenicity was predicted to be 0.8909 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. To prevent possible allergies, allergy prediction was performed on the linked polypeptide II using an AllerTOP v.2.0 on-line tool, and as a result the protein was not allergic. The protein is subjected to three-dimensional modeling by using a ZpGJn4 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 (see figure 1).

The amino acid sequence of the final polypeptide II multi-epitope vaccine is as follows:

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 subjected to lactic acid bacteria codon preference optimization, and the base sequence of the finally determined multi-epitope peptide fragment is as follows:

GCTAAATTTGTTGCTGCTTGGACATTAAAAGCTGCTGCTGAAGCTGCTGCTAAATGG

AAAGTTGTTACAGGTTTAAATGGTTTTGGTAATGTTTTAAATGATTTAACAAATGGTGGTA

CAGGTAAAAAATTAGGTCGTACAAAAGAAGCTTTTGCTACACCAGTTACAGGTGGTGTTGA

TGGTATTCCACATATTGGTTTTTCAGAATATGAAGGTGGTTGTGTTGTTGTTCGTAAACCA

GATGGTCAAACAAATAAAAAAGGTGGTAAAAAAGCTTTAGGTATTGCTAATGGTCAAACAA

ATGAAGCTACATTTAATCAAGCTGTTACAACATCAACACAATGGTCAGCTCCATTAAATTT

AGCTATTGGTAAAAAAGTTAATGGTAATCGTACATCAACAATTGATTTAGGTCAAGCTGCT

ATTTCAGGTCATGGTACAGTTGTTGATTTTAAATTAAAACCAGCTCCAGGTTCAAATGATT

GTTTAGCTAAAACAAATGCTCGTATTGATTGGTCAGGTTCAATGAATTCATTAGGTTTTAA

TAATACAGCTTCAGGTAATACAGCTGCTAAAGGTGGTAAAAAAGATGGTTTATCATCAATT

TTTTATGGTGGTTTACCACGTGGTTCAGAATTATCAGCTGGTTCAGCTGCTGCTGCTCGTA

CAAAATTATTTGGTTCATTATCACGTGATGATATTTTAGGTCAAATTCAACGTGTTAATGC

TAATAATACATCAGCTGCTTATCGTGCTACAAATGTTGGTAATGGTTCAGGTGGTGCTAAT

ATTAATACATCATTTACAACAGCTGAATATACACATACATCAGCTATTCAATCATTTAATT

ATTCAGCTCAATTAAAAAAAGATGATCGTGCTCCATCAAATGGTGGTTATAAAGCTGGTGT

TTTTACAACATCAGCTTCAGCTGCTTATAAATTAACAATTACAGTTACAGGTAATAAAGCT

GCTTATGTTGTTGATTTTAAATTAAAACCAGCTCCAGGTTCAAATGATTGTTTACCAGGTC

CAGGTGCTTATTTTGTTTTACCAATGAAAAATGCTGAAGGTACAAAATAA(SEQ ID NO：2)。

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

Example 2: construction of recombinant lactococcus lactis

1. Amplification of the target fragment.

The pET28a-XDT plasmid is used as a template, and XDT-F, XDT-R is used as a primer to amplify the signal peptide sequence. Plasmid extraction kit is used to extract plasmid from bacterial strain pET28a-KT, and KT-F, KT-R primer is used as template to amplify the nucleic acid sequence of multivalent epitope peptide. 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, ddH2O 7. 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 9: PCR primer sequence table for amplifying target fragment

2. Extraction and restriction enzyme digestion of pNZ8149 plasmid

pNZ8149/NZ3900 strain (laboratory preservation) strainInoculating to GM17 medium, standing at 30deg.C overnight, collecting 2mL of bacterial liquid, and extracting pNZ8149 plasmid by using gram positive bacterial plasmid small extraction kit (Solarbio). Using restriction enzymes SacI and NcoI, the system was 2. Mu.L NEBbuffer, 8. Mu.L pNZ8149 plasmid, 1. Mu.L Sac1, 1. Mu.L Nco1, 8. Mu.L ddH ₂ 0. After heating in a 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 the subsequent carrier construction.

3. Construction of recombinant lactic acid bacteria strains

The amplified XDT sequence fragment and KT sequence fragment were recovered by a gel recovery kit, mixed with the double digested and purified pNZ8149 plasmid fragment, and added with a seamless cloning PreMix Premix (Monad) 5.5. Mu. L, XDT fragment 1.5. Mu. L, KT fragment 1.5. Mu.L and the recovered pNZ8149 plasmid 2. Mu.L, followed by water bath at 37℃for 25min. Mixing 5 μl of the above connection product with 50 μl of LNZ3900 competent cells, incubating on ice for 15min, transferring to a shock cup (1 mm) pre-cooled for 15min, 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-KT, and the sequence of the recombinant plasmid is correct through sequencing. The construction of the pNZ8149-KT/NZ3900 strain was confirmed to be successful.

4. Stability test of recombinant strains

The recombinant strain pNZ8149-KT/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 98% of the recombinant strain remained stable after 20 passages (see FIG. 2).

The stability test result of the recombinant strain pNZ8149-KT/NZ3900 shows that the stability of the pNZ8149-KT/NZ3900 strain is basically consistent with that of the parent strain pNZ8149/NZ3900, and more than 98% of positive clones still exist after passage for 20 generations.

Taking empty fungus as a control group and recombinant fungus as a test group, inoculating the empty fungus to M17OD was measured every half hour in (0.5% lactose) medium ₆₀₀ Drawing and comparing the growth curves of the two, and finding that the growth curves of the recombinant bacteria and the parent bacteria are basically consistent (see figure 3); 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.

The growth curve of the recombinant strain shows that the growth curve of the pNZ8149-KT/NZ3900 strain is basically consistent with that of the parent strain pNZ8149/NZ3900, and no obvious difference exists.

5. Preparation of lactobacillus material for immunization

The identified lactobacillus pNZ8149-KT/NZ3900 is inoculated into 5mLGM17 liquid culture medium and is statically cultivated at a 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 NZ8149-KT/NZ3900 lactobacillusAnd (3) quantitatively feeding immune NZ8149-KT/NZ3900 bacteria material to the bacteria material test group. 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; mice were boosted a third time at 59d, 60d, 61d, 62d, 63d for 5 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 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, igA antibodies of mice in the experimental group are found to be remarkably reduced in OD value compared with a control group (see figure 4), and the remarkable increase of IgA antibodies of the mice in the experimental group is proved (judged according to the kit specification); the K88 antibody and the K99 antibody of the experimental group were also significantly elevated compared to the control group, and the differences were extremely significant (see fig. 5, 6).

Meanwhile, amino acid sequences of the connecting polypeptide I and the connecting polypeptide III are utilized to be translated into nucleotide sequences in a reverse direction, lactococcus lactis capable of expressing the connecting polypeptide I and the connecting polypeptide III is constructed, mice are immunized, and as a result, the connecting polypeptide I and the connecting polypeptide III strain stimulate the K88 antibody and the K99 antibody produced by the mice, the titers of the connecting polypeptide I and the connecting polypeptide III strain are lower than those of the connecting polypeptide II strain stimulated 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 that predicted by software.

Because the diarrhea-causing escherichia coli mainly infects animals through the digestive tract, severe damage to intestinal mucosa can be caused, obvious increase of the secretory IgA antibody, the K88 antibody and the K99 antibody of the experimental group indicates that the diarrhea-causing escherichia coli can effectively prevent the damage to the intestinal tract, reduce the infection caused by the intestinal tract, and lay a solid foundation for successful development of oral vaccine for the diarrhea of the clinical pig escherichia coli.

Claims (10)

1. A fusion polypeptide, wherein 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 carrying the gene of claim 3.

5. The recombinant expression vector of claim 4, wherein the recombinant expression vector is a pNZ8149 plasmid expression vector.

6. A recombinant strain comprising the fusion polypeptide of claim 1.

7. The recombinant strain of claim 6, wherein the recombinant strain is lactococcus lactis transformed with the recombinant expression vector of claim 4.

8. Use of the fusion polypeptide of claim 1 for the preparation of a swine enterotoxigenic escherichia coli subunit vaccine.

9. Use of the recombinant strain according to claim 6 or 7 for the preparation of live vaccine of enterotoxigenic escherichia coli in pigs.

10. A vaccine comprising the fusion polypeptide of claim 1 and/or the recombinant strain of claim 6 or 7.