CN115819625A - Tetravalent antigen fusion polypeptide of escherichia coli - Google Patents
Tetravalent antigen fusion polypeptide of escherichia coli Download PDFInfo
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Abstract
The invention provides an escherichia coli tetravalent antigen fusion polypeptide, and 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 above fusion polypeptide. Compared with a control group, the content of secretory IgA antibody, K88, K99, 987P, F antibody of the recombinant strain serving as a vaccine immune animal is obviously increased. The prepared fungus material can protect most of catharsis escherichia coli causing pig diarrhea.
Description
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 (also called cocktail vaccine) is a subunit vaccine designed based on the amino acid sequence of the target antigen epitope. The multi-epitope vaccine can be divided into linear tandem multi-epitope vaccine, multivalent antigen peptide epitope vaccine, virus particle-like vaccine and the like. According to different antigen epitope types, the vaccine can be divided into B cell epitope vaccine, T cell epitope vaccine, mixed epitope vaccine and the like.
Colibacillosis diarrhea is an infectious disease with extremely high morbidity and mortality caused by enterotoxigenic escherichia coli, and brings serious economic loss to the pig raising industry. The piglets have the symptoms of diarrhea, dehydration, slow growth and the like, and even die suddenly. Meanwhile, the immunity of the piglets is low, and the piglets are easy to infect other bacteria, viruses and the like, so that the normal development of the live pigs is influenced. Colibacillosis has very obvious age characteristics, and can be divided into 3 types of piglet yellow dysentery, piglet white dysentery and piglet edema disease according to the age difference of the diseased days. The yellow scour of piglets occurs within 7 days of age, and has the characteristics of high morbidity and high mortality; the white scour of piglets occurs at the age of 10-20 days, and has the characteristics of high morbidity and low mortality; the edema disease of the piglet 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 frequently generated in cold winter and hot and humid summer. The incidence and death rate of the disease are influenced by various factors such as the immune state of the swinery, climate change, feeding management level, growth environment and the like. In order to ensure normal growth and marketing of live pigs, a plurality of farms and free-ranging farmers regularly use antibiotic medicines in the live pig breeding process
The disease prevention and treatment of the drugs can cause the antibiotic drugs to remain in the live pigs after long-term use or excessive use, which not only affects the quality of pork products, but also destroys the soil and ecological environment due to the antibiotic contained in the excrement and urine excretion. At the same time, long-term use of large amounts of antibiotics leads to increased resistance in pathogenic bacteria. Escherichia coli of porcine origin has been resistant to 19 antibiotics, and the efficacy of antibiotics is being 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 carriers has attracted more and more attention. The lactobacillus oral vaccine can be directly orally taken without injection administration, thereby avoiding the risk of injury and infection caused by injection; can imitate natural infection path, induce mucosal immune response, and activate immune system; has immunological adjuvant effect, and the surface of lactobacillus can improve immunity and enhance vaccine effect.
The basic principle of vaccine immunization is to utilize biological agents which can stimulate immune response reaction without causing harm, so that the body obtains corresponding immunity, and the immune response reaction is not directed against the whole foreign substance, but is directed against epitope, usually a section of polypeptide. The epitope vaccine is a novel vaccine developed in recent years, which is used as a vaccine by expressing or artificially synthesizing the epitope of pathogenic microorganisms in vitro by using a genetic engineering means. The epitope is also called an antigenic determinant, is a chemical group determining the specificity of antigen in an antigen molecule, and is a basic unit capable of being specifically combined with a T cell antigen receptor TCR or a B cell antigen receptor BCR, and finally stimulates the immune response of an organism to form the immunity to pathogenic microorganisms. According to the difference of epitopes, epitope vaccines are divided into B epitope vaccines, T epitope vaccines and multi-epitope vaccines with two epitopes. The multi-epitope vaccine can induce B lymphocytes to generate antibodies, can activate cytotoxic lymphocytes to generate cellular immune response, and has better application prospect.
Disclosure of Invention
The invention aims to provide an escherichia coli tetravalent antigen fusion polypeptide, wherein the antigen fusion polypeptide is prepared by fusing antigen epitope peptides screened from escherichia coli K88, K99, 987P and F18 antigen genes, and the fusion polypeptide can efficiently express the antigen epitopes of the four genes in lactococcus lactis, so that the recombinant lactococcus lactis for efficiently and recombinantly expressing the fusion polypeptide is provided.
The invention provides an escherichia coli tetravalent antigen fusion polypeptide, which comprises the following components:
1) A polypeptide having the amino acid sequence of SEQ ID NO. 1;
2) Polypeptide derived from 1) by substituting, deleting and adding one or more amino groups on the sequence of 1);
the invention also provides a gene, wherein the gene codes the fusion polypeptide, and one nucleotide sequence of the gene is SEQ ID NO. 2;
in still another aspect, the present invention provides a recombinant expression vector, wherein a gene segment encoding the fusion polypeptide is inserted into the recombinant expression vector;
the invention also provides a recombinant strain 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 the preparation of vaccines.
In still another 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 a vaccine to immunize animals, and the content of secretory IgA antibody, K88, K99, 987P, F antibody is obviously increased compared with that of a control group. The prepared bacterial material can protect most of catharsis escherichia coli (K88, K99, 987P, F) causing pig diarrhea.
Drawings
FIG. 1 is a three-dimensional model diagram of a tetravalent polyepitope fusion protein of Escherichia coli,
FIG. 2: graph of IgA antibody detection results of immunized mice, wherein IgA antibody detection results of immunized mice: p is less than 0.01, and the difference is extremely obvious; in the graph, the OD value of IgA antibody detection of the pNZ8149-8991/NZ3900 strain experimental group is obviously reduced compared with that of the pNZ8149/NZ3900 lactobacillus control group and a blank control group, and the IgA antibody of mice in the experimental group is obviously improved and is extremely different;
FIG. 3: graph of the results of the detection of K88, K99, 987P, F antibodies in immunized mice, wherein: p is less than 0.01, and the difference is extremely obvious; in the figure, the OD values detected by K88, K99 and 987P, F18 antibodies of the pNZ8149-8991/NZ3900 strain experimental group are obviously increased compared with those of the pNZ8149/NZ3900 lactobacillus control group and the blank control group, and the difference is extremely obvious.
Detailed Description
The adhesin is an important virulence factor of pathogenic escherichia coli and can be attached 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 porcine escherichia coli pili that, once colonized in 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 respectively 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 K88, K99, 987P and F18 genes are directly coupled to form a coupled protein, the expression efficiency in lactococcus lactis is low. The invention carries out molecular sequence analysis on cell epitopes of Escherichia coli K88, K99, 987P and F18, couples selected epitope polypeptides to form fusion polypeptides, clones a nucleic acid fragment coded by the fusion polypeptides into an Escherichia coli-lactobacillus shuttle plasmid pNZ8149, and electrically converts the fusion polypeptides into lactococcus lactis NZ3900 to prepare a recombinant strain. The recombinant strain immunized mice are used for preparing clinical candidate vaccines of escherichia coli.
The present invention will be described in detail below with reference to specific embodiments and the accompanying 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 are respectively carried out on Escherichia coli K88, K99, 987P and F18 genes.
(1) Analytical screening of B cell epitope polypeptides
B cell epitope of K88ac gene of Escherichia coli K88, K99, 987P and F18 gene is predicted by using IEDB and ABCPred online tools. The target fragment is a fragment whose prediction results coincide with each other, preferably a sequence having a long fragment, and adjacent sequences are combined. Finally, 12 fragments are selected as the amino acid fragments of the Escherichia coli B cell epitope, and the related information is shown in Table 1.
Table 1: finally determined Escherichia coli B cell epitope polypeptide information table
(2) Prediction of Th cell epitopes
The Th cell epitope of K88, K99, 987P and 18F protein is predicted, and the amino acid sequence of the Th cell epitope peptide of K88 is AYFVLPMKNAEGTK (named as K88-Th-CTL-129-142), and the amino acid sequence of the Th cell epitope peptide of K99 is VVDFKLKPAPGSNDCL (named as K99-Th-43-58). While the Th cell epitope peptide of 987P is coincided with 987P-B-36-88, and the Th cell epitope peptide of F18 protein is coincided with F18-B-95-144.
(3) Prediction of CTL cell epitopes
CTL epitopes were predicted for K88, K99, 987P and 18F proteins. Wherein, no dominant epitope exists in the IEDB database; in NetMHC-4.0, molecular binding peptides HLA-A2, HLA-A0201, HLA-A0202, HLA-A0203, and HLA-A0205 were selected, respectively, and the predicted overlapping sequences of the molecular binding peptides were integrated usingbase:Sub>A neural network and quantization matrix method (ANN + QM) withbase:Sub>A threshold of 0.5 to obtain 2 CTL epitopes (Table 2).
Table 2: CTL epitope prediction results table for K88, K99, 987P and 18F
| Serial number | Initiation site | Peptide fragment | Naming or remarking |
| 1 | 69 | KLTITVTGNK | K88-CTL-69-78 |
| 2 | 120 | |
987P-CTL-120-132 |
Example 2: construction and screening of tetravalent antigen fusion polypeptide
And constructing and screening fusion polypeptides from the determined polyepitope peptide fragments of the K88, K99, 987P and F18 genes.
1. Construction of fusion polypeptide I
The 16 fragments are arranged according to the sequence of 1 Th cell epitope, 3B cell epitopes, 2B-CTL cell epitopes, 1 CTL cell epitope, 1 Th cell epitope, 3B cell epitopes, 1B-CTL cell epitope, 1B Th CTL cell epitope, 1B cell epitope and 1B Th 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, and 987P-B-Th-TCL-36-88.
Adding a general Th cell epitope (AKFVAAWTLKAAA) to the N end of the sequence, wherein EAAAK is an N-end connector, PGPG is a Th cell epitope connector, GKK is a B cell epitope connector, and AAY is a CTL cell epitope connector. The antigenicity of the antigen is predicted to be 0.9856 by using VaxiJen online 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.
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。
a universal Th cell epitope (AKFVAAWTLKAAA) is added to the N-terminal of the sequence, and a linker sequence is connected with the polypeptide I. The antigenicity of the vaccine is predicted to be 0.9885 by using VaxiJen online software.
3. Fusion polypeptide III
The 16 fragments were arranged in the order of K88-K99 cell epitopes (B cell epitope, B cell-CTL cell fusion epitope, CTL cell epitope, th cell-CTL cell fusion epitope) and 987P-F18 cell epitopes (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.
A universal Th cell epitope (AKFVAAWTLKAAA) is added at the N-terminal of the sequence, EAAAK is an N-terminal connector, and the connector sequence is connected with the polypeptide I. The antigenicity of the vaccine is predicted to be 0.9822 by using VaxiJen online software.
Comparing the 3 linked polypeptides, the antigenic property of linked polypeptide II is the strongest, and the antigenic property of linked polypeptide III is the weakest. The protein has no allergy as a result of the allergy prediction of the linker polypeptide II using the AllerTOP v.2.0 online tool. The protein is subjected to three-dimensional modeling by using an I-TASSER online tool, the structural epitope is better exposed and is easy to combine with an antibody, and the epitope design requirement is met on the protein molecular conformation (figure 1); the figure shows that the protein domains interfere with each other less, the surface-located domains are abundant, and the structural epitope exposure is better.
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 a Th universal cell epitope site, and the underlined part is a Linker.
The lactobacillus codon preference optimization is carried out on the amino acid of the multi-epitope vaccine, and the finally determined base sequence of the 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 sequence was synthesized by Shanghai Biotechnology engineering, inc., and cloned into pET28a plasmid, which was designated as pET28a-8991.
Example 3: construction of recombinant lactococcus lactis
1. And (3) amplification of the target fragment.
pET28a-XDT plasmid was used as a template, and XDT-F, XDT-R was used as a primer to amplify a signal peptide sequence (the sequence of the primer is shown in Table 3). Extracting from bacterial strain pET28a-8991 by using plasmid extraction kitThe nucleotide sequence of the multivalent epitope peptide was amplified using the plasmid as a template, primers 8991-F and 8991-R (see Table 3 for primer sequences). The CR reaction system and conditions were as follows: the PCR reaction system is 20 μ L, including 2 XTAQA Master Mix 10 μ L, primer F1 μ L, primer R1 μ L, vector μ L, ddH 2 O7. Mu.L. Reaction procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 1min, annealing at 46 ℃ for 1min, extension at 72 ℃ for 90s, and 35 cycles in total; final extension at 72 ℃ for 10min.
Table 3: PCR primer sequence table for target fragment amplification
Extraction and restriction of pNZ8149 plasmid
pNZ8149/NZ3900 strain (stored in the laboratory) is inoculated into GM17 medium, and is kept standing and cultured overnight at 30 ℃, 2mL of bacterial liquid is taken, and the pNZ8149 plasmid is extracted by using a gram-positive bacterium plasmid miniextraction kit (Solarbio). The restriction enzymes Sac I and Nco I are used for double digestion, and the digestion systems are 2 mu L NEB buffer, 8 mu L LpNZ8149 plasmid, 1 mu L Sac1, 1 mu L Nco1 and 8 mu L ddH 2 0. Heating in water bath at 37 ℃ for 3h, detecting the enzyme digestion product by agarose gel electrophoresis, and recovering and purifying a target band by a gel recovery kit (Omega) for vector construction.
3. Construction of recombinant lactic acid bacteria strains
The XDT sequence fragment and the 8991 sequence fragment amplified above were recovered with a gel recovery kit, mixed with the pNZ8149 plasmid fragment after double digestion and purification, added with 1.5. Mu.L of 5. Mu. L, XDT fragment PreMix (Monad), 1.5. Mu.L of 8991 fragment and 2. Mu.L of recovered pNZ8149 plasmid, and subjected to water bath at 37 ℃ for 25min. And mixing 5 mu L of the ligation product with 50 mu L of NZ3900 competent cells, placing the mixture on ice for incubation for 15min, transferring the mixture into a precooled electric shock cup (1 mm) for 15min, carrying out 1250V electric shock, then quickly adding 950 mu L of resuscitation medium, and acting for 1.5-2h at 30 ℃. Inoculating 100 mu L of the recombinant plasmid into an ELIKER plate, culturing for 2 days at 30 ℃, selecting a yellow single colony to 5mL of liquid culture medium, standing and culturing overnight at 30 ℃, extracting a recombinant plasmid pNZ8149-8991 by using a positive bacterium plasmid extraction kit, and sequencing the recombinant plasmid to show that the sequence is correct. The success of construction of pNZ8149-8991/NZ3900 strain was confirmed.
4. Stability test of recombinant strains
The recombinant strain pNZ8149-8991/NZ3900 is passaged in an M17 (0.5% lactose) culture medium, the strain is inoculated in an Eliker screening culture medium after 20 generations, more than 98% of colonies are still positive colonies, and the plasmids are extracted and are identified to be correct through sequencing. It was shown that more than 97% of the recombinant strains remained stable after 20 passages (see table 4).
Table 4: positive colony ratio table (unit: 100%) of parent and recombinant strain after passage
| Generation (generation) | 0 | 5 | 10 | 15 | 20 |
| pNZ8149/NZ3900 | 100 | 99 | 99 | 98 | 98 |
| pNZ8149-8991/NZ3900 | 100 | 98 | 98 | 97 | 97 |
The airborne bacteria are used as a control group, the recombinant bacteria are used as a test group, the control group and the recombinant bacteria are inoculated in an M17 (0.5% lactose) culture medium, and OD is measured every half an hour 600 (see table 5), 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; meanwhile, the growth curves of the two are not obviously different by t test. The growth performance of the recombinant bacteria is proved to be unaffected compared with that of the parent bacteria.
Table 5: OD of parent strain and recombinant strain in different growth periods 600 Value table
5. Preparation of lactobacillus material for immunization
Inoculating identified lactobacillus pNZ8149-8991/NZ3900 into 5mLGM17 liquid culture medium, and culturing at constant temperature of 30 ℃ for overnight. The next day, 5mL of the activated bacterial solution was inoculated into 200mL of LGM17 medium for further culture. Cultured to OD 600 When the concentration is 0.3-0.4, adding Nisin solution with the final concentration of 10ng/mL, standing and inducing for 6 hours at 30 ℃, and then mixing the bacterial solution 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 concentration of 200mL/L, mixing well, and dripping into CaCl with concentration of 2% 2 And (3) generating gel beads in the solution, filtering, washing with pure water for 3 times, and solidifying and shaping for 30-60 min. Filtering, washing with pure water for 3 times, adding into 1% chitosan solution (with lactobacillus ratio of 1:1), stirring, coating with film 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 M17 liquid culture medium, standing for 10min, homogenizing with a beating type homogenizer, and counting by using a plate colony counting method (or turbidimetry) after homogenizing. According to the counting result, the concentration of the original bacteria liquid is adjusted to ensure that the immune concentration of the bacteria material is 5 multiplied by 10 9 cfu/g, mice were immunized by quantitative feeding.
18 BABL/c mice were randomly divided into 3 groups A-F, each group was 6. Wherein the first group was set as a blank control, and was not immunized with any vaccine; the second group is set as lactobacillus strain control, and quantitatively feeding immune empty carrier pNZ8149/NZ3900 lactobacillus strain material; the third group is NZ8149-8991/NZ3900 lactobacillus material test group, and the immune NZ8149-8991/NZ3900 lactobacillus material is quantitatively fed. The immunization program is as follows: priming mice at 1d, 2d, 3d, 4d, 5d for 5 days of continuous immunization; immunizing mice for 2 times at 30d, 31d, 32d, 33d and 34d for 5 days continuously; mice were given a third booster immunization at 59d, 60d, 61d, 62d, 63d for 5 consecutive days. The immunization dose is as follows: quantitative feeding of 5X 10 per mouse 9 cfu/g bacterial material 2g. 25 days after the last immunization, all 18 mice were sacrificed and serum was collected; and cutting the small intestine of the ileum section by 5cm, washing the intestinal mucosa with 200 μ L of intestinal lavage fluid, and collecting intestinal fluid.
Serum and intestinal lavage fluid samples are detected by referring to the specifications of a mouse K88 antibody ELISA detection kit, a K99 antibody ELISA detection kit, a 987P antibody ELISA detection kit, an F18 antibody ELISA detection kit and a secretory IgA antibody ELISA detection kit. The IgA antibody ELISA detection kit is a competitive ELISA kit, and through detection, the IgA antibody of an experimental group mouse is found, compared with a control group, the OD value is obviously reduced (shown in figure 2), and the IgA antibody of the experimental group mouse is proved to be obviously increased; the K88 antibody, K99 antibody, 987P antibody and F18 antibody in the experimental group were also significantly increased compared to the control group (see fig. 3).
Meanwhile, the laboratory reversely translates the amino acid sequence of the connecting polypeptide into a nucleotide sequence, clones upstream and downstream sequences by a company, clones and expresses the connecting polypeptide by using lactococcus lactis, and immunizes a mouse, so that the K88 antibody generated by the mouse stimulated by the connecting polypeptide I and the connecting polypeptide III is lower in titer than the antibody generated by the mouse stimulated by the connecting polypeptide II, the result is proved to be higher than the antigenicity of the connecting polypeptide I and the connecting polypeptide III, and the result is consistent with the antigenicity result predicted by software. The diarrhea-causing escherichia coli infects animals mainly through the digestive tract, and can cause serious damage to the intestinal mucosa.
Claims (10)
1. An escherichia coli tetravalent antigen fusion polypeptide, wherein said fusion polypeptide comprises:
1) A polypeptide having the amino acid sequence of SEQ ID NO. 1;
2) Polypeptide which is derived from the 1) by substituting, deleting and adding one or a plurality of amino groups on the sequence of the 1).
2. A gene encoding the fusion polypeptide of claim 1.
3. The gene of 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/transfected with the recombinant expression vector of claim 4.
6. The recombinant strain of claim 5, wherein the recombinant strain is recombinant lactococcus lactis.
7. Use of the fusion polypeptide of claim 1 in the preparation of a vaccine.
8. Use of the recombinant strain of claim 5 for the preparation of a vaccine.
9. A vaccine, wherein an antigen of said vaccine comprises the fusion polypeptide of claim 1.
10. The vaccine of claim 1, wherein said antigen further comprises the recombinant strain of claim 5.
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| US20040087522A1 (en) * | 1999-12-27 | 2004-05-06 | Ronald Marquardt | Genetic vaccines for the production of chicken egg-yolk antibodies against enterotoxigenic escherichia coli and other pathogens |
| WO2007010040A1 (en) * | 2005-07-22 | 2007-01-25 | Novoplant Gmbh | Antigen binding polypeptides against f4 (k88) fimbriae |
| CN103275228A (en) * | 2013-06-05 | 2013-09-04 | 黑龙江八一农垦大学 | K99-987P-F41 recombinant protein and application thereof |
| CN105061602A (en) * | 2015-08-03 | 2015-11-18 | 江苏省农业科学院 | Fusion protein for detecting anti-ETEC (enterotoxigenic escherichia coil) antibody of pigs, as well as preparation method and application thereof |
| CN109293753A (en) * | 2018-09-29 | 2019-02-01 | 赛法特（长沙）生物技术有限公司 | A kind of enterotoxigenicEscherichia coli tetravalence dynein immunogene and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040087522A1 (en) * | 1999-12-27 | 2004-05-06 | Ronald Marquardt | Genetic vaccines for the production of chicken egg-yolk antibodies against enterotoxigenic escherichia coli and other pathogens |
| WO2007010040A1 (en) * | 2005-07-22 | 2007-01-25 | Novoplant Gmbh | Antigen binding polypeptides against f4 (k88) fimbriae |
| CN103275228A (en) * | 2013-06-05 | 2013-09-04 | 黑龙江八一农垦大学 | K99-987P-F41 recombinant protein and application thereof |
| CN105061602A (en) * | 2015-08-03 | 2015-11-18 | 江苏省农业科学院 | Fusion protein for detecting anti-ETEC (enterotoxigenic escherichia coil) antibody of pigs, as well as preparation method and application thereof |
| CN109293753A (en) * | 2018-09-29 | 2019-02-01 | 赛法特（长沙）生物技术有限公司 | A kind of enterotoxigenicEscherichia coli tetravalence dynein immunogene and preparation method thereof |
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