Toxicity weakened NSP4 mutant gene, recombinant plasmid and recombinant bovine rotavirus

A technology for mutating genes and recombining plasmids, applied in genetic engineering, plant genetic improvement, viruses/bacteriophages, etc., can solve problems such as short duration

Active Publication Date: 2014-05-07
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

NSP4 protein and its short peptides synthesized in vitro can cause diarrhea in animals, with clinical symptoms similar to intact RV virulence, but with a shorter duration (Zhang et al., 2000)

Method used

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  • Toxicity weakened NSP4 mutant gene, recombinant plasmid and recombinant bovine rotavirus
  • Toxicity weakened NSP4 mutant gene, recombinant plasmid and recombinant bovine rotavirus
  • Toxicity weakened NSP4 mutant gene, recombinant plasmid and recombinant bovine rotavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1: Rescue bovine rotavirus with mutation of NSP4 gene virulence site by reverse genetic technology

[0079] 1 Construction of recombinant plasmids with mutations in NSP4-shRNA recognition sites

[0080] Our laboratory has isolated a strain of bovine rotavirus G6, and delivered the identified NSP4 gene sequence to GenBanK. Its coding sequence (FJ972713.1) is shown in SEQ ID NO.1. The strain of bovine rotavirus G6 is Regular wild-type bovine rotavirus. Referring to its sequence, using the siRNA design software provided by Applied Biosystems, the target gene sequence of RNAi was determined to be 5'-TGAACAGCACATTGCACAC-3' (as shown in SEQ ID NO.20), and its recognition site on the NSP4 gene is Areas 91-109. The pEASY-T3-NSP4 recombinant plasmid constructed by conventional methods was used as a template, and under the guidance of primers RNAi-P1, RNAi-P2, RNAi-P3, and RNAi-P4 (see Table 1), respectively, PCR amplification was carried out. The reaction conditions we...

Embodiment 2

[0089] Example 2: Screening and Identification of Rescued Viruses

[0090] 1 Construction of shRNA recombinant lentiviral plasmid targeting NSP4 gene

[0091] After annealing chemically synthesized NSP4-shRNA-1 and NSP4-shRNA-2, LacZ-shRNA-1 and LacZ-shRNA-2 (see Table 3, as shown in SEQ ID NO.11, 12, 13, and 14) respectively , and the 12.5kb fragment recovered after double digestion with XbaI and AgeI of the lentiviral H1 vector and the 1.5kb fragment obtained by double digestion with SmaI and AgeI ( figure 2) according to the following system: 12.5kb vector fragment, 100ng; 1.5kb vector fragment, 50ng; shRNAoligo, 50ng; T4DNA ligase, 0.5μL; 10×T4DNA ligase buffer, 2μL, supplemented with H 2 O Bring the system to 20 μL and ligate overnight at 16°C. The competent cells of Escherichia coli DH5α strain were transformed, clones were selected, plasmid DNA was extracted, and the corresponding shRNA recombinant H1 vector was obtained by PCR identification and sequencing, which wa...

Embodiment 3

[0114] Embodiment 3: the virulence identification of mutant virus BRV-AABY

[0115] Determination of virus titer of 1BRV-AABY

[0116] Freeze and thaw the mutant virus BRV-AABY and wild-type BRV three times to fully release the virus particles, centrifuge at 5000r / min at 4°C for 10-15min, make 10-fold serial dilutions with serum-free DMEM, and take 10 -1 ~10 -8 The diluent is ready for use. Well-grown MA104 cells were treated with 2×10 4 ~3×10 4 Cells / ml were inoculated into a 96-well cell culture plate, and the culture medium was discarded after 24 hours, and the virus was inoculated from the highest dilution with a pipette, and each dilution was inoculated into 8 wells, 100 μl per well. MA104 cells inoculated with virus were incubated at 37°C, 5% CO 2 Cultivate in an incubator, observe cell lesions under a light microscope after 72 hours, and calculate BRV-AABY and BRV TCID by ReedMuench method 50 .

[0117] 2 Animal infection experiment

[0118] Thirty-three clean-g...

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Abstract

The invention discloses a toxicity weakened NSP4 mutant gene, and the nucleotide sequence of the NSP4 mutant gene is shown as SEQ ID No.2 in a sequence table. The invention further discloses a recombinant plasmid containing the toxicity weakened NSP4 mutant gene and a recombinant bovine rotavirus containing the toxicity weakened NSP4 mutant gene, the construction method is that cotransfection of rhesus nephrocyte MA104 is carried out on the recombinant plasmid and a pcDNA3.1-T7RNAP recombinant plasmid, then a wide bovine rotavirus is inoculated, and a reverse inheritance technique is used for saving heteromorphosis virus to obtain toxicity weakened recombinant bovine rotavirus. The recombinant bovine rotavirus firstly uses the inheritance technique to obtain toxicity weakened BRV viral strains at home and abroad, compared with a traditional method for obtaining attenuated strains, the toxicity weakened NSP4 mutant gene uses the reverse inheritance technique and combines with an RNA interference method, successfully obtains the toxicity weakened bovine rotavirus, and provides a rapid path for obtaining BRV attenuated vaccine candidate strains.

Description

technical field [0001] The present invention relates to NSP4 mutant gene with weakened virulence, recombinant plasmid and recombinant bovine rotavirus (bovine rotavirus, BRV), and its construction method, especially relates to a bovine rotavirus obtained by using reverse genetics technology (Reverse Genetics Technology). The method for virus attenuation strain belongs to the technical field of genetic engineering. Background technique [0002] Bovine rotavirus (BRV) belongs to the genus Rotavirus in the Reoviridae family and is one of the main pathogens of non-bacterial diarrhea. It mainly infects calves aged 1 to 7 days. It has the characteristics of wide prevalence, high incidence, and great harm. It has become a worldwide disease. BRV infection can easily cause calf digestive tract dysfunction, secondary bacterial diarrhea, and then aggravate the disease, resulting in increased calf mortality and reduced production performance of recovered calves, seriously hindering the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/46C12N15/85C12N15/66C12N7/01C12R1/93
Inventor 何洪彬宋玲玲杨宏军王洪梅
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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