Bovine viral diarrhea virus BVDV-BJ175170 and application thereof

A BVDV-BJ175170, bovine viral diarrhea technology, applied in the direction of application, virus, virus peptide, etc., can solve the problems of long delivery period, undetectable antibody, high price, etc., to reduce the missed detection rate, high sensitivity, The effect of high precision

Inactive Publication Date: 2021-02-12
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the late start of research on BVDV in my country, insufficient attention has been paid to the awareness and hazards of BVDV, and no systematic nationwide molecular epidemiological survey of BVDV has been carried out. Research data; at the same time, BVDV is an RNA virus and is prone to genetic mutations. It is difficult to prepare detection kits and vaccines with traditional standard strains to deal with all existing clinical strains
On the other hand, currently imported BVDV detection kits are expensive, and there is no self-developed commercial BVDV antibody detection kit in China, which makes it difficult to realize large-scale cattle herd epidemiological screening and population purification on large-scale cattle farms
[0004] At present, the commercially available IDEXX Bovine Viral Diarrhea Virus Antibody Detection Kit has a long delivery time and is expensive; other recombinant proteins are used as the coating antigen, and E2 protein is mostly used. Although E2 protein is the most important protein that stimulates the production of antibodies in animals. , but its conservation is low, the protein is easy to mutate, and the virus in the BVDV epidemic area mutates and cannot detect antibodies

Method used

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  • Bovine viral diarrhea virus BVDV-BJ175170 and application thereof
  • Bovine viral diarrhea virus BVDV-BJ175170 and application thereof
  • Bovine viral diarrhea virus BVDV-BJ175170 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 BVDV clinical strain isolation identification and pathogenicity research

[0060] 1. Sample source

[0061] In November 2017, a blood sample was collected from a cow farm suspected of BVDV in Beijing, and the serum was collected after low-temperature centrifugation.

[0062] 2. Sample BVDV antigen ELISA kit detection

[0063] ① Add 50 μL of detection buffer to the first 4 reaction wells of the detection plate.

[0064] ② Load 50 μL of the negative control, positive control, and serum samples to be tested (No. 175170).

[0065] ③ Mix the buffer solution and the sample to be tested in the detection plate by vibrating with an oscillator, and incubate at 37°C for 2 hours.

[0066] ④ Discard the liquid in the detection plate, add 300 μL of diluted Wash Solution to rinse 5 times.

[0067] ⑤ Add 100 μL enzyme-labeled antibody, place at room temperature for 30 minutes, and rinse 5 times.

[0068] ⑥ Add 100 μL of TMB substrate solution, incubate in dark room at ro...

Embodiment 2

[0169] Example 2 pET-32a-E rns Construction of prokaryotic expression plasmids

[0170] 1. Primer design and synthesis, designed primers to amplify E rns Genes and primers were designed as follows:

[0171] F: 5′-CCC GGATCC ATGGAAAACATAACACAGTGGAACCTAC-3′ (with Bam HI restriction site)

[0172] R: 5′-CCC CTCGAG TTAAGCGTATGCTCCAAACCACG-3′ (with Xho I restriction site)

[0173] 2. Amplification of the target gene: PCR amplification is performed using the reverse transcription product of bovine viral diarrhea virus as a template. PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 62°C for 30 s, extension at 72°C for 100 s, a total of 30 cycles; final extension at 72°C for 10 min. Amplified E rns Gene fragments were recovered using a PCR product purification kit.

[0174] After PCR amplification, identified by agarose gel electrophoresis, the results are shown in Figure 8 , the position of the band is correct, whic...

Embodiment 3

[0179] Prokaryotic expression and purification of embodiment 3 recombinant protein

[0180] 1. For the colony pET-32a-E that is sequenced correctly in Example 2 rns , pick a single colony and inoculate + In liquid LB medium, shake overnight at 37°C. Take the overnight culture and inoculate it with 2% of Amp-containing + In the liquid LB medium, shake culture at 37°C until A 600The value is 0.5-1.0, add IPTG to the final concentration of 1mM, shake culture at 30°C and 37°C respectively, and take 1mL bacterial liquid from the culture at different time of culture (0, 3, 6, 10h) and transfer to centrifuge In the tube, centrifuge at 10000r / min for 5min, collect the bacterial pellet, resuspend the pellet, ultrasonically break it, centrifuge at 4000r / min for 10min, take 10μL of supernatant and pellet, add 90μL of 2× loading buffer and 10μL of 1mol / L DTT suspension mix Evenly, boil for 5min, carry out SDS-PAGE electrophoresis (8% separating gel, 5% stacking gel), to determine the ...

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Abstract

The invention provides a bovine viral diarrhea virus BVDV-BJ175170 and application of the bovine viral diarrhea virus BVDV-BJ175170, wherein the preservation number of the BVDV-BJ175170 is CGMCC NO. 18339. According to the invention, closing and expressing of the E<rns> protein of the bovine viral diarrhea virus BVDV-BJ175170 is used for ELISA detection of BVDV and preparation of a kit; the kit disclosed by the invention is convenient to operate, low in cost and good in repeatability, can be used for detecting the viral diarrhea virus antibody of which the serum dilution reaches 1:6400, overcomes the defect of lack of a single antigen for BVDV-PI cattle diagnosis at present, has relatively high sensitivity, reduces the omission ratio, is relatively high in precision, and can be used for antibody level monitoring; and according to the method, the high-specificity reaction of the antigen and the antibody is combined, the E<rns> protein antibody in serum can be detected more accurately, asensitive detection means is provided for BVDV epidemiological investigation in China, and the method can be used for early diagnosis of bovine viral diarrhea virus infection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to bovine viral diarrhea virus BVDV-BJ175170 and application thereof. Background technique [0002] Bovine Viral Diarrhea Virus (BVDV), also known as Bovine Viral Diarrhea-Mucosa Virus, can present a variety of clinical symptoms in infected cattle, such as cough, fever, diarrhea, mucosal ulcers or erosions, etc. The performance of leukopenia, persistent infection, immunosuppression and tolerance, abortion, malformation or stillbirth of pregnant cows. Since Lafson first discovered the disease in New York State of the United States in 1946, it has been widely prevalent in the world. It not only infects cattle, but also infects goats, sheep, deer and other ruminants, causing serious economic losses to animal husbandry around the world. Epidemiological survey results show that BVDV presents a trend of widespread infection in Beijing, and persistent infection of cattle is the main cause of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C07K14/18C12N15/40C07K16/10G01N33/569A61K39/12A61P31/14C12R1/93
CPCC12N7/00C07K14/005C07K16/1081G01N33/56983A61K39/12A61P31/14C12N2770/24321C12N2770/24034C12N2770/24322G01N2333/18G01N2469/20C12N2770/24334A61K2039/552A61K2039/5252
Inventor 王九峰王胜华杨仁杰聂嘉伟丁成志杨光辉朱要宏颜晓冬
Owner CHINA AGRI UNIV
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