Porcine rotavirus VP4 truncated protein and its expression and application in preventing and treating porcine diarrhea

Abstract

The present application provides a truncated protein of porcine rotavirus VP4 and its expression and application in preventing and treating porcine diarrhea; the amino acid sequence of the truncated protein of porcine rotavirus VP4 of the present application is SEQ ID NO.3, which has improved immunogenicity by appropriately truncating the nitrogen-terminal part of VP4 and introducing histidine and proline mutations; and can be used to prepare injectable / oral vaccines or drugs.

Description

Technical Field

[0001] The present application belongs to the field of proteins and immunity. Specifically, the present application provides a truncated protein of porcine rotavirus VP4 and its expression and application in preventing and treating porcine diarrhea. Background Art

[0002] Porcine rotavirus (PoRV) can induce acute diarrhea and dehydration in young pigs, and is one of the common causes of death / poor growth in young pigs. Currently, there are no effective drugs or vaccines for porcine rotavirus (only a few whole virus inactivated vaccines are under research), and in farming practice, it can generally only be controlled by disinfection, isolation and other means.

[0003] VP4 protein is a protein known to have neutralizing activity / potential in porcine rotavirus, and some studies have used this protein to prepare injectable or oral vaccines. However, the application for VP4 protein is relatively large, and there are deficiencies in expression, production and safety. Therefore, many studies have used truncated proteins of this protein, which are deficient in inducing immunity and often fail to produce effective protection or therapeutic effects in practice. Summary of the invention

[0004] Based on the previously studied VP4 truncated protein, the applicant borrowed the stabilization methods of other vaccines and attempted to introduce proline and histidine mutations to improve the stability of the protein, thereby improving the immune effect of the truncated protein.

[0005] On the one hand, the present application provides a porcine rotavirus VP4 truncated protein, the amino acid sequence of the porcine rotavirus VP4 truncated protein being SEQ ID NO.3.

[0006] On the other hand, the present application provides a gene encoding the above-mentioned porcine rotavirus VP4 truncated protein, and the gene sequence is SEQ ID NO.4.

[0007] On the other hand, the present application provides a host, which expresses the gene sequence shown in SEQ ID NO.4.

[0008] Furthermore, the host is Escherichia coli.

[0009] On the other hand, the present application provides the use of the above-mentioned porcine rotavirus VP4 truncated protein or host in the preparation of a method for treating or preventing porcine rotavirus infection.

[0010] On the other hand, the present application provides a drug for treating or preventing porcine rotavirus infection, wherein the drug comprises the above-mentioned porcine rotavirus VP4 truncated protein or host.

[0011] Furthermore, the drug is in the form of an injection or oral administration.

[0012] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0013] On the other hand, the present application provides a method for preparing the above-mentioned porcine rotavirus VP4 truncated protein, which comprises expressing the gene sequence shown in SEQ ID NO.4 in a host.

[0014] Furthermore, the host is Escherichia coli.

[0015] Furthermore, the method also includes a purification step.

[0016] Beneficial effects:

[0017] The VP4 truncated protein of the present application is of suitable size and can be easily purified by prokaryotic expression cassette. After the introduction of proline and histidine mutations, the immune performance is significantly improved compared with the initial truncated protein or similar truncated proteins, providing a good material for the development of vaccines and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the electrophoresis diagram of the gene amplification and protein expression results of the VP4 truncated protein of the present application;

[0019] Figure 2 The IgG levels in the serum of mice after immunization with three VP4 truncated proteins are shown;

[0020] Figure 3 Shown are the levels of neutralizing antibodies in mouse serum after mice were immunized with three VP4 truncated proteins. DETAILED DESCRIPTION

[0021] Example 1 Design, expression and purification of VP4 truncated protein

[0022] Based on the prediction of secondary structure by SOPMA, a proline mutation and a histidine mutation (SEQ ID NO.3, corresponding to the gene sequence SEQ ID NO.4) were introduced into the random coil part of the existing VP4 truncated protein (SEQ ID NO.1 previously used by the applicant, corresponding to the gene sequence SEQ ID NO.2) to improve stability and thus immunogenicity. The specific sequence is shown below and in the sequence listing file:

[0023] SEQ ID NO.1:

[0024] MASLIYRQLLTNSYTVDLSDEIQEIGSTKSQNVTINPGPFAQTGYAPVNWGPGEINDSTTVEPLLDGPYQPTTFNPPVDYWMLLAPTTPGVIVEGTNNTDRWLATI LI EPNVQSENRTYTI FGIQEQLTVSNTSQDQWKFIDVVKTTANGSIGQYGPLLSSPKLYAVMKHNEKLYTYEGQTPNARTAHYSTTNYDSVNMTAFCDFYI IPRSEESKCTEYINNGLPPIQNTRNVVPLSLTARDVI HYRAQANEDIVIS

[0025] SEQ ID NO.2:

[0026] Atggcgagcctgatctaccgtcagctgctgaccaactcttacaccgttgacctgagcgatgaaatccaggaaatcggcagcaccaaaagccagaacgttaccatcaacccaggtccgttcgctcagaccggctacgcgccggttaactggggtccgggtgaaattaacgatagcaccaccgttgaaccgctgctggatggtccgtaccagccgaccaccttcaacccgccggttgattactggatgctgctggcgccgaccactccgggcgttatcgttgaaggtaccaacaacaccgatcgttggctggcgaccatcctgatcgaaccgaacgttcagagcgaaaaccgtacctacaccatcttcggcatccaggaacagctgaccgtttctaacacctctcaggatcagtggaaattcatcgatgttgtgaaaaccaccgcgaacggttctatcggccagtatggtccgctgctgtccagcccgaaactgtacgcggttatgaaacacaacgaaaaactgtacacctacgaaggtcagaccccgaacgcgcgtaccgcgcactacagcaccaccaactacgattccgttaacatgaccgcgttctgcgatttctacatcatcccgcgtagcgaagaaagcaaatgcaccgaatacatcaacaacggcctgccgccgatccagaacacccgtaacgttgttccgctgagcctgaccgcgcgtgatgttatccactaccgtgcgcaggcgaacgaagatatcgttatcagc

[0027] SEQ ID NO.3:

[0028] MASLIYRQLLTNSYTVDLSDEIQEIGSTKSQNVTINPGPFAQPGYAPVNWGPGEINDSTTVEPLLDGPYQPTTFNPPVDYWMLLAPTTPGVIVEGTNNTDRWLATI LI EPNVQSENRTYTI FGIQEQLTVSNTSHDQWKFIDVVKTTANGSIGQYGPLLSSPKLYAVMKHNEKLYTYEGQTPNARTAHYSTTNYDSVNMTAFCDFYI IPRSEESKCTEYINNGLPPIQNTRNVVPLSLTARDVI HYRAQANEDIVIS

[0029] SEQ ID NO.4:

[0030] Atggcgagcctgatctaccgtcagctgctgaccaactcttacaccgttgacctgagcgatgaaatccaggaaatcggcagtaccaaaagccagaacgttaccatcaacccaggtccgttcgctcagccaggctacgcgccggttaactggggtccgggtgaaattaacgatagcaccaccgttgaaccgctgctggatggtccgtaccagccgaccaccttcaacccgccggttgattactggatgctgctggcgccgaccactccgggcgttatcgttgaaggtaccaacaacaccgatcgttggctggcgaccatcctgatcgaaccgaacgttcagagcgaaaaccgtacctacaccatcttcggcatccaggaacagctgaccgtttctaatacctctcacgatcagtggaaattcatcgatgttgtgaaaaccaccgcgaacggttctatcggccagtatggtccgctgctgtccagcccgaaactgtacgcggttatgaaacacaacgaaaaactgtacacctacgaaggtcagaccccgaacgcgcgtaccgcgcactacagcaccaccaactacgattccgttaacatgaccgcgttctgcgatttctacatcatcccgcgtagcgaagaaagcaaatgcaccgaatacatcaacaacggcctgccgccgatccagaacacccgtaacgttgttccgctgagcctgaccgcgcgtgatgttatccactaccgtgcgcaggcgaacgaagatatcgttatcagc

[0031] The VP4 truncated protein was prepared basically according to the description in the prior application:

[0032] Entrust Jin Si Te to artificially synthesize the gene sequence (see Figure 1Left part); the gene sequence and pET32a plasmid were double digested with BamH I and Xho I, the digestion products were separated by agarose gel electrophoresis, and the target band and the vector band were recovered by gel cutting; T4 ligase was used for ligation, and the cells were transformed into BL21 (DE3) competent cells, and 0.5ug / ml ampicillin was used for screening to obtain recombinant bacteria carrying the plasmid of the new VP4 truncated protein gene; a single colony grown on the plate was picked for pure culture and the plasmid was extracted and sequenced for identification.

[0033] The identified correct recombinant bacteria were cultured in LB liquid medium containing 0.5ug / ml ampicillin at 37°C for 12 hours under shaking conditions; the bacterial solution cultured overnight under shaking conditions was inoculated into LB medium to make its initial OD600 concentration 0.2; the culture was cultured at 37°C under shaking conditions for about 2 hours, and when the OD600 reached about 0.6, an IPTG inducer with a final concentration of 1mM was added; the fermentation was terminated after 8 hours of induction, and the bacterial solution was taken.

[0034] The VP4 truncated protein was purified using an affinity chromatography nickel column (Shanghai Yansheng), and the protein expression was detected by SDS-PAGE gel electrophoresis ( Figure 1 The purified VP4 truncated protein was obtained (right part), and the protein concentration was determined using a Bradford protein concentration kit for subsequent immunization work.

[0035] Example 2 Mouse Immunization Experiment

[0036] Forty BALB / C female mice (6 weeks old, 25±2 g) purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. were randomly divided into four groups and used in the experiment after 10 days of adaptive training.

[0037] On Day 0, purified VP4 truncated protein (SEQ ID NO.1), VP4 truncated protein (SEQ ID NO.3), VP8 (amino acids 1-241 of VP4) and PBS were mixed with MF59 adjuvant and then immunized mice at a dose of 50 μg / mouse (multiple injections on the back).

[0038] Blood samples were collected on Day 28, Day 35, and Day 42:

[0039] The IgG antibody level was detected using the mouse IgG double antibody sandwich ELISA detection kit from Shanghai Jiya Biotechnology Co., Ltd.; G9P7 strain.

[0040] Rotavirus (G9P7 strain) was diluted to 100TCID50 / 50 μL using DMEM medium; mouse serum collected was graded diluted using DMEM medium; serum and virus solution were mixed in equal volumes; the mixture was added to the wells of the Vero cell monolayer (100 μL / well) and incubated at 37 degrees Celsius for 2 hours; the mixture was discarded, washed, cultured and the pathological conditions were observed; and the neutralizing antibody level was calculated.

[0041] The results are as follows Figure 2 and Figure 3 As shown: In terms of IgG, there is no significant difference between the three VP4 truncated proteins. Within 6 weeks, the IgG level increased over time. In terms of neutralizing antibody titers, at around 5 weeks, there was little difference between SEQ ID NO.1 and SEQ ID NO.3, both were better than VP8. At 6 weeks, SEQ ID NO.3 significantly exceeded SEQ ID NO.1 due to improved stability and other reasons.

[0042] Example 3 Actual pig farm use effect

[0043] The recombinant bacteria in Example 1 were cultured on a large scale to form bacterial powder, which was added to pig feed at a ratio of 0.3%. The pig feed was used to feed pigs with typical symptoms of porcine rotavirus (diarrhea, aversion to solids, vomiting, etc., and porcine rotavirus infection was identified by diagnostic reagents and identification). The results showed that after eating the feed containing bacterial powder, the symptoms of most pigs were alleviated, and the feed intake and weight gain basically returned to normal within 2 weeks.

Claims

1. A porcine rotavirus VP4 truncated protein, characterized in that: The amino acid sequence of the porcine rotavirus VP4 truncated protein is SEQ ID NO.

3.

2. The gene encoding the porcine rotavirus VP4 truncated protein according to claim 1, characterized in that: The gene sequence is SEQ ID NO.

4.

3. A host, characterized in that The host expresses the gene sequence shown in SEQ ID NO.4, and the host is Escherichia coli.

4. Use of the porcine rotavirus VP4 truncated protein according to claim 1 or the host according to claim 3 in the preparation of a drug for treating or preventing porcine rotavirus infection.

5. A drug for treating or preventing porcine rotavirus infection, characterized in that: The drug comprises the porcine rotavirus VP4 truncated protein according to claim 1 or the host according to claim 3.

6. The drug according to claim 5, wherein the drug comprising the porcine rotavirus VP4 truncated protein according to claim 1 is in the form of an injection; and the drug comprising the host according to claim 3 is in the form of an oral dosage form.

7. The drug according to claim 6, further comprising a pharmaceutically acceptable excipient.

8. The method for preparing the porcine rotavirus VP4 truncated protein according to claim 1, characterized in that: The method comprises expressing the gene sequence shown in SEQ ID NO.4 in a host; the host is Escherichia coli.

9. The method according to claim 8, further comprising a purification step.

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