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Caprine arthritis encephalitis virus-encephalitis virus serum antibody diagnosis labelled protein gp135 and preparation method thereof

An encephalitis virus and serum antibody technology, applied in the biological field, can solve problems such as unfavorable industrial production, differences in protein secondary and tertiary structures, and large differences between protein expression systems and eukaryotic cells, and achieve convenient industrial production. good effect

Inactive Publication Date: 2017-12-19
新疆畜牧科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the recombinant expression method of gp135 protein is mainly prokaryotic expression represented by Escherichia coli expression. CAEV virus infects mammalian epithelial cells, and the protein expression system of the host is used for translation and expression, while the protein expression system of prokaryotic cells is quite different from that of eukaryotic cells. Large, due to the difference in codon preference and the difference in the protein structure formation environment, the expressed protein has a large difference in amino acid sequence, secondary and tertiary structure; in addition, E. coli expression has formed inclusion bodies, which is not conducive to industrial production

Method used

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  • Caprine arthritis encephalitis virus-encephalitis virus serum antibody diagnosis labelled protein gp135 and preparation method thereof
  • Caprine arthritis encephalitis virus-encephalitis virus serum antibody diagnosis labelled protein gp135 and preparation method thereof
  • Caprine arthritis encephalitis virus-encephalitis virus serum antibody diagnosis labelled protein gp135 and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, optimization and synthesis of CAEV gp135 gene

[0021] (1) Homology analysis of CAEV gp135 gene

[0022] Homology analysis was performed on the gene encoding gp135 protein of three strains of goat arthritis-encephalitis virus (CAEV) isolated in my country (GeneBank: KT749879.1, KT749880.1, KT749881.1) published in GeneBank. Sequence comparison analysis shows that the gp135 gene sequences encoded by the three CAEV strains have small differences, with a homology of more than 99.2%, and a homology of more than 78% with the gene sequences published internationally, see figure 1 .

[0023] (2) Codon optimization of CAEV gp135 gene

[0024] According to the codon preference in the expression protein of Pichia pastoris GS115, the stability of the induced expression protein, glycosylation, formation of secreted expression, etc., the gp135 protein coding gene was optimized, and the synthesis department of Shanghai Bioengineering Technology Co., Ltd. was entruste...

specific example 1

[0035] Specific example 1. Diagnosis of CAEV-infected sheep by Western blot

[0036] After goats were infected with goat arthritis-encephalitis virus (CAEV), they were stimulated to produce antibodies. The gp135 recombinant protein expressed and purified by gp135 / GS115 engineering bacteria was used as an antigen to detect the serum antibodies of the goats tested by Western Blotting. It can be used for serological diagnosis of CAE.

[0037] 1. Material method

[0038] 1.1 Materials

[0039] Serum: 3 CAEV-infected goat serum samples, including 2 positive samples and 1 negative sample;

[0040] Antigen: gp135 protein induced by gp135 / GS115 engineering bacteria;

[0041] Secondary antibody: HRP-labeled rabbit anti-goat IgG (Tiangen Biotechnology Co., Ltd.).

[0042] 1.2 Test method

[0043] According to "Appendix 8 Common Techniques in Molecular Cloning - Western Blotting" (p 1723-1726) in the third edition of "Molecular Cloning-Experimental Guide" published by Science Press:...

example 2

[0051] Example 2. Diagnosis of CAEV-infected sheep by agarose gel diffusion method

[0052] Goats were infected with goat arthritis-encephalitis virus (CAEV) and stimulated to produce antibodies. The gp135 recombinant protein expressed and purified by gp135 / GS115 engineering bacteria was used as an antigen, and the goat serum tested was detected by agarose gel diffusion method (AGID). Antibodies can be used for serological diagnosis of CAE.

[0053] 1.1 Materials

[0054] Serum: 10 CAEV-infected goat serum samples, including 3 positive samples and 6 negative samples;

[0055] Antigen: 80 KD gp135 protein induced, expressed and purified by gp135 / GS115 engineering bacteria;

[0056] 1.2 Test method

[0057] Refer to "SNT 1171.2-2003 Goat Arthritis-Encephalitis Antibody Detection Method-Agar Immunodiffusion Test", gp135 / GS115 engineered bacteria induced expression, purified recombinant gp135 protein 2μg / well.

[0058] 2. Results

[0059] The results of AGID showed that the g...

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Abstract

The invention belongs to the technical field of biology, and concretely relates to caprine arthritis encephalitis virus-encephalitis virus serum antibody diagnosis labelled protein gp135. The caprine arthritis encephalitis virus-encephalitis virus serum antibody diagnosis labeled protein gp135 contains an optimized gp135 protein nucleotide sequence shown as SEQ ID NO.1 and the other sequences with consistency being not less than 95%; and contains the optimized gp135 protein nucleotide sequence shown as SEQ ID NO.2 and the other sequences with consistency being not less than 95%. The gp135 diagnosis labelled protein expressed through pichia yeast GS115 has good reactogenicity and is convenient for industrial production, and provides antigen for developing a CAEV high flux serological diagnosis method.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a goat arthritis-encephalitis virus serum antibody diagnostic marker protein gp135 and a preparation method thereof. Background technique [0002] Caprine arthritis-encephalitis (CAE) is a chronic viral infectious disease of goats caused by caprine arthritis-encephalitis virus (CAEV). The main features of the disease are: adult goats show slowly developing arthritis, sometimes accompanied by interstitial pneumonia and interstitial mastitis; 2-6 month old lambs show neurological symptoms of ascending paralysis. The disease has a high infection rate and a long incubation period. Infected goats carry the virus for life, and there is no specific treatment method. It has an important impact on the production performance of the herd, especially the milk production, and also hinders the normal trade of breeding animals. It is listed in my country. It is a second-class animal dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/155C12N15/49C12N15/81G01N33/569C12R1/84
CPCC07K14/005C12N15/815C12N2740/15022C12N2800/22G01N33/56983G01N2333/155G01N2469/20
Inventor 吴建勇王登峰李建军古努尔·吐尔逊刘志强杨学云金映红刘艳丰班万里王文奇马晓菁叶锋刘丽娅蒋晓梅
Owner 新疆畜牧科学院
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