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Construction and application of recombinant lactobacillus paracasei displaying porcine rotavirus VP7 protein on surfaces

A technology of Lactobacillus casei-like and porcine rotavirus, which is applied in the field of construction of recombinant Lactobacillus casei-like, can solve the problems of difficulty in prevention and control and the like

Pending Publication Date: 2019-07-23
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The first purpose of the present invention is to provide a recombinant Lactobacillus casei that displays the VP7 protein of porcine rotavirus on the surface, so as to solve the problem that rotavirus infects animals through mucous membranes and is difficult to prevent and control

Method used

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  • Construction and application of recombinant lactobacillus paracasei displaying porcine rotavirus VP7 protein on surfaces
  • Construction and application of recombinant lactobacillus paracasei displaying porcine rotavirus VP7 protein on surfaces
  • Construction and application of recombinant lactobacillus paracasei displaying porcine rotavirus VP7 protein on surfaces

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This example illustrates the construction of recombinant Lactobacillus paracasei.

[0041] 1. Obtaining the target gene.

[0042] According to the full gene sequence of PoRV VP7 published in GeneBank, the accession number is GenBank: AB180971.1, a pair of specific primers were designed by using DNA Man, and the restriction sites of SalI and HindIII were introduced at the 5' ends of the upstream and downstream primers respectively. Protective bases (Table 1, underlined enzyme cleavage sites). Using the cDNA of porcine rotavirus BNCC124944 as a template, the target fragment was amplified by PCR. PCR reaction conditions: pre-denaturation at 95°C for 5 minutes; (94°C for 1 minute, annealing at 58°C for 1 minute, and 72°C for 2 minutes), a total of 30 cycles; final extension at 72°C for 10 minutes. PCR products were analyzed on a 0.9% agarose gel. The full length of PoRV VP7 was amplified to about 1062bp ( figure 1 ).

[0043] Table 1 PCR primer sequences

[0044]

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Embodiment 2

[0050] This example illustrates the identification of the expression of pLA-VP7 / E.coli.

[0051] 1. SDS-PAGE detection

[0052] Correctly identified positive recombinant lactic acid bacteria pLA-VP7 and pLA empty bacteria were expanded and cultivated. Take 2h, 4h, 6h, 8h, 10h of recombinant lactic acid bacteria and 10h of pLA / E.coli bacteria, ultrasonically crushed, and carried out SDS- PAGE, the results showed that the relative molecular mass of the expressed recombinant VP7 protein was about 82kDa ( image 3 ), which is consistent with the expected value.

[0053] Second, Western blot identification

[0054] The protein samples of recombinant bacteria pLA-VP7 / E.coli and pLA / E.coli expressed for 6 hours were subjected to SDS-PAGE, and the protein in the gel was transferred to PVDF membrane, and the membrane was blocked with 5% skimmed milk for 2 hours. Discard the skim milk, wash with PBST three times, wash the skim milk on the membrane and discard the PBST, use the porcin...

Embodiment 3

[0056] This example illustrates the identification of the expression of pLA-VP7 / L.paracasei.

[0057] 1. Indirect immunofluorescence identification

[0058] Take 300 μL of recombinant bacteria pLA-VP7 / L.paracasei and pLA / L.paracasei expressed for 6 hours, centrifuge at 5000r / min for 5min, discard the supernatant and keep the bacteria, resuspend the bacteria in 1mL PBS, centrifuge for 5min, discard the supernatant , this step is repeated 3 times. Finally, dilute the bacteria with PBS solution, take out the glass slide soaked in 75% ethanol and dry it naturally, add 10 μL of diluted sample (depending on the number of bacteria) on the glass slide, let it stand at room temperature until the sample is air-dried, and put the glass slide Soak the slices in ice-cold absolute ethanol for 30 minutes to fix the bacteria, slowly wash off the absolute ethanol on the glass slides with sterile water, and then put them in 5% skimmed milk for overnight sealing; take out the slides the next da...

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Abstract

The invention relates to recombinant lactobacillus paracasei displaying porcine rotavirus VP7 protein on surfaces. The recombinant lactobacillus paracasei displaying porcine rotavirus VP7 protein on surfaces has the gene sequence inserting porcine rotavirus VP7 protein in a lactobacillus paracasei genome. The invention also provides a construction method and an application of the recombinant lactobacillus paracasei. Through obtaining the whole-length gene sequence of the porcine rotavirus VP7, the recombinant lactobacillus paracasei is constructed, and mice and piggies are immunized in an oraltaking manner; and the immune effects of the recombinant strain are evaluated, the result shows that the immune effects of an antigen epitope are reinforced, and a feasibility basis is provided for research of preparation of orally-taken rotavirus genetic engineering lactic acid bacteria.

Description

technical field [0001] The invention belongs to the field of animal molecular biology and relates to the construction and application of a recombinant lactobacillus paracasei which displays porcine rotavirus VP7 protein on the surface. Background technique [0002] Porcine rotavirus (PoRV for short) infection mostly occurs in the cold season and is an endemic infectious disease. Sick pigs are the main source of infection. Antibodies to PoRV can be found in the feces of infected pigs within 5 to 10 days. It is transmitted through the fecal-oral route. Most pigs are recessively infected. Piglets aged 15 to 35 days are susceptible. Pigs infected with rotavirus can produce circulating antibodies under natural conditions. Sows adopt rotavirus antibodies to piglets through breast milk. The piglets are allowed to acquire maternal antibodies for 3 to 5 weeks. Compared with the piglets of multiparous sows, the piglets of primiparous sows are more likely to be infected with porcine ro...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74A61K39/15A61P31/14C12R1/225
CPCC07K14/005C12N15/74A61K39/15A61P31/14C12N2720/12322
Inventor 余丽芸李丽阳侯喜林
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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