Schistosoma japonicum katsurada recombinant protein and its preparation method and use

A technology of recombinant protein and schistosomiasis, which is applied in the field of bioengineering, can solve the problems of no public reports of diagnostic antigens and achieve good application value

Inactive Publication Date: 2016-05-11
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Our laboratory discovered the Schistosoma japonicum vesicular membrane protein SjVamp2 in the research on the surface membrane proteomics of Schistosoma japonicum, but its important role in the growth and development of Schistos...

Method used

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  • Schistosoma japonicum katsurada recombinant protein and its preparation method and use
  • Schistosoma japonicum katsurada recombinant protein and its preparation method and use
  • Schistosoma japonicum katsurada recombinant protein and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Expression and purification of Schistosoma japonicum recombinant protein

[0041] 1. Method

[0042] 1.1 Construction of recombinant expression plasmids

[0043] According to the amino acid sequence of SjVamp2 with the NCBI accession number gb|AAP05935.1|, the gene sequence of the open reading frame of the coding region was found, and primers were designed. .3); downstream primer P2: 5'-GTTCTCGAGTCACTGAGTAGCACTTCCA-3' (containing XhoI restriction site, SEQ ID NO.4), used to amplify the SjVamp2 gene sequence. Using the cDNA of Schistosoma japonicum 42-day-old body as a template, PCR amplifies its cDNA fragment containing ORF, and the reaction composition is as follows:

[0044]

[0045] PCR reaction conditions: pre-denaturation at 94°C for 5min, denaturation at 94°C for 30sec, annealing at 56.5°C for 30sec, extension at 72°C for 1min, a total of 30 cycles; the final cycle at 72°C for 10min. After the PCR product was purified, it was connected to the pMD19...

Embodiment 2

[0054] Example 2 Detection of Antigenicity and Immunogenicity of Schistosoma japonicum Recombinant Protein rSjVamp2

[0055] 1. Western Blotting analysis of antigenicity and immunogenicity of recombinant protein

[0056] Carry out SDS-PAGE electrophoresis with the purified rSjVamp2 protein, and then transfer it to the NC membrane at 4°C with 130mA for 1h, block overnight, and then use the specific antibody serum of rSjVamp2 protein and the whole worm immune rabbit serum as primary antibodies Perform westernblot to analyze the antigenicity and immunogenicity of rSjVamp2 protein.

[0057] 2. The results of Western Blot analysis of the antigenicity and immunogenicity of the recombinant protein

[0058] The results of Western blot showed that when the above two sera were used as primary antibodies, a relatively obvious band appeared at the size of 21kD, indicating that the rSjVamp2 protein has good antigenicity and immunogenicity. image 3 The results of western blot using the s...

Embodiment 3

[0059] Example 3 Transcript level analysis of Schistosoma japonicum SjVamp2

[0060] 1. Method

[0061] 1.1 Real-time quantitative PCR analysis of the expression of SjVamp2 in different developmental stages of Schistosoma japonicum

[0062] Using Schistosoma japonicum α-Tublin as an internal reference gene, the upstream amplification primer of real-time quantitative PCR is 5'-CTGATTTTCCATTCGTTTG-3'(SEQ ID NO.5); the downstream primer is 5'-GTTGTCTACCATGAAGGCA-3'(SEQ ID NO.6). The upstream amplification primer for SjVamp2 real-time quantitative PCR is 5'-ACAACCTCGACCACAGAACAAG-3' (SEQ ID NO.7), and the downstream primer is 5'-TTCCTGCACTAGCCTCGAATTG-3' (SEQ ID NO.8).

[0063] The cercariae, 7, 14, 21, 28, 35, and 42-day-old worm bodies of Schistosoma japonicum, as well as 42-day-old female and male worm bodies and eggs were collected, and the total RNA of each stage was extracted. Using the PrimeScriptRTreagentKitWithgDNAEraser (TaKaRa) kit, the genomic DNA in the RNA sample w...

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Abstract

The invention discloses a schistosoma japonicum katsurada recombinant protein. The schistosoma japonicum katsurada recombinant protein is prepared through expression of a schistosoma japonicum katsurada Vamp2 gene-containing recombinant vector. The invention also discloses a use of the schistosoma japonicum katsurada recombinant protein as a schistosoma japonicum katsurada diagnosis antigen and a use of the schistosoma japonicum katsurada recombinant protein in preparation of a vaccine or drug for preventing or treating bilharziasis. In a mouse immunization experiment, the schistosoma japonicum katsurada recombinant protein can induce generation of anti-recombinant protein specific IgG, IgG1 and IgG2a antibodies in a mouse and an antibody generation level is high. An animal protection experiment result shows that the recombinant protein has a potential as an anti-bilharziasis candidate vaccine and a novel drug target. A diagnosis antigen effect assessment experiment result shows that the recombinant protein has a potential as a diagnosis antigen and has a wide application prospect.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant protein of Schistosoma japonicum and its preparation method and application. Background technique [0002] Schistosomiasis is a major parasitic disease that seriously endangers the health of humans and animals. It is prevalent in 76 countries and regions around the world. About 650 million people are threatened by infection, 200 million people are infected, and the annual death toll exceeds 100,000. Schistosomiasis japonicum is prevalent in our country. Although my country has made great achievements in the prevention and control of schistosomiasis, due to the difficulty of eliminating the intermediate host snails, the variety of terminal hosts, the wide range of activities, and the difficulty in controlling the source of infection, the phenomenon of repeated infection of schistosomiasis is serious, and there is immune escape. The prospects for controlling th...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/70A61K39/00A61P33/12G01N33/68C07K16/18
CPCY02A50/30
Inventor 林矫矫傅志强韩倩曹晓丹洪炀陆珂刘艳涛马帅马茜茜陆看吕超王涛宰金丽贾秉光张祖航
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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