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CA-TM fusion protein and application of CA-TM fusion protein in detection of Medi-Vass virus

A fusion protein and virus technology, applied in the biological field, to achieve the effect of broad market prospects, high sensitivity and strong specificity

Pending Publication Date: 2022-04-29
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently only adequate control procedures are in place to limit the spread of the virus or to eradicate the disease

Method used

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  • CA-TM fusion protein and application of CA-TM fusion protein in detection of Medi-Vass virus
  • CA-TM fusion protein and application of CA-TM fusion protein in detection of Medi-Vass virus
  • CA-TM fusion protein and application of CA-TM fusion protein in detection of Medi-Vass virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] The acquisition of embodiment 1CA-TM fusion protein

[0086] 1. Acquisition of CA-TM fusion gene

[0087] According to the whole gene (NM1111) of the Inner Mongolia strain of sheep Madedi-Visna Virus (MVV) that has been registered in NCBI, the accession number (GI: MW248464) selects the highly antigenic and conserved positions of the CA gene and the TM gene The primers for amplifying the CA gene and the TM gene were designed using Primer software, and were synthesized by Shanghai Sangon Bioengineering Co., Ltd. Among them, the upstream primer CA-F-1 of the CA gene: 5'-ATGCAAGCAGGAGGAAGAAGTTGG-3', as shown in SEQ ID NO.2; the downstream primer CA-R-645L containing the linker: 5'- agaaccgcctcctcc AAATCCTTCTGAGCCCACATCT-3' (linker is underlined), as shown in SEQ ID NO.3. TM gene and upstream primer containing linker GP-F-1L: 5'- ggaggaggcggttct CTTGCTAACGCTACTGCCGC-3' (linker is underlined), as shown in SEQ ID NO.4; downstream primer GP46-R-405: 5'-CTACCATGAGAACCATGTT...

Embodiment 2

[0097] Example 2 Establishment of Meddy-Visna virus antibody indirect ELISA detection method and kit

[0098] 1. Screening of optimal antigen coating concentration and serum dilution multiple

[0099] Specific steps are as follows:

[0100] 1) Coating antigen: use the purified CA-TM fusion protein as the coating antigen, dilute it with the coating solution (set the antigen coating concentration as 5μg / 100uL, 1μg / 100uL, 0.5μg / 100uL, 0.25μg / 100uL , 0.1 μg / 100uL total of 5 concentration gradients) and placed in the microtiter plate respectively, 100 μL / well, covered the microtiter plate with a cover film, and incubated overnight at 4°C for coating.

[0101] 2) Blocking: Add blocking solution, 200 μL / well, cover the plate with a cover film, incubate at 37°C for 1 hour, discard the reaction solution, add 200 μL washing solution to each well, and wash 4 times;

[0102] 3) Serum addition: use the sample diluent to dilute the negative control serum and the positive control serum by ...

Embodiment 3

[0174] Embodiment 3 Meddy-Visna virus antibody indirect ELISA detection method detects the sensitivity test of different coating antigens

[0175] The Medi-Visna virus antibody indirect ELISA detection method established with embodiment 2 uses CA protein, TM protein and CA-TM fusion protein as coating antigen respectively under the same conditions to the same positive serum (positive serum is respectively 200 times, 400-fold, 800-fold, 1600-fold, 3200-fold and 6400-fold dilution times) for detection and evaluation of the sensitivity of the detection method.

[0176] The result is as Figure 11 As shown in Table 4, the results show that: when the CA-TM fusion protein is detected as a coating antigen (expressed as CA-TM ELISA in the figure and table), the minimum can be detected as positive after 3200 times of diluted serum, and the CA protein is used as a coating antigen. When it is detected by antigen (indicated by CA ELISA in the figure and table) and when TM protein is used...

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Abstract

The invention discloses a CA-TM fusion protein and an application of the CA-TM fusion protein in detection of a Medi-Vass virus. The invention firstly discloses a CA-TM fusion protein with an amino acid sequence as shown in SEQ ID NO. 1. The invention further discloses an indirect enzyme-linked immunosorbent assay (ELISA) detection kit and a detection method for the Midde-Wisla virus antibody, wherein the indirect ELISA detection kit comprises an elisa plate taking the CA-TM fusion protein as a coating antigen. The indirect enzyme-linked immunosorbent assay (ELISA) detection method for the Mede-Vass virus antibody is established on the basis of the CA-TM fusion protein, is used for detecting the level of the Mede-Vass virus antibody, has the characteristics of strong specificity, high sensitivity, good repeatability, convenience in operation and the like, has a high coincidence rate with the existing international commercial kit in clinical detection, can realize large-scale detection, and has a wide application prospect. And an effective means is provided for serological detection and epidemiological investigation of the Medi-Visner disease, and the kit has a relatively wide market prospect.

Description

technical field [0001] The present invention relates to the field of biotechnology. More specifically, it relates to a CA-TM fusion protein and its application in detecting Maedi-Visna virus. Background technique [0002] Maedi-Visna Disease (MVD) is a slow, contact and progressive inflammatory disease in sheep caused by Maedi-Visna Virus (MVV). The length of the MVV genome is between 9200 nucleotides, mainly composed of the main genes (gag, pol and env) and regulatory genes (vif, tat and rev) common to the three retroviruses. The coding genes correspond to structural proteins (Gag, Pol, and Env) and regulatory proteins (Vif, Tat, and Rev), respectively. gag encodes internal structural proteins that protect DNA, the largest of which is the capsid protein (Capsid protein, CA, also known as p25), which stimulates the host to produce antibodies and is often used in ELISA detection methods. env encodes the following two proteins inserted into the envelope: surface glycoprotei...

Claims

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Application Information

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IPC IPC(8): C07K19/00G01N33/569G01N33/543G01N33/58G01N33/535
CPCC07K14/005G01N33/56983G01N33/54306G01N33/581G01N33/535C07K2319/00C12N2740/10022G01N2333/15G01N2469/20Y02A50/30
Inventor 刘淑英齐景伟陈思旭
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY