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A kind of Schistosoma japonicum recombinant protein sjsaplp5 and its coding gene and application

A recombinant protein, schistosomiasis technology, applied in recombinant DNA technology, application, genetic engineering and other directions, to achieve the effect of good immunogenicity and good antigenicity

Active Publication Date: 2018-08-28
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity (90%) of the diagnostic kit still has room for further improvement

Method used

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  • A kind of Schistosoma japonicum recombinant protein sjsaplp5 and its coding gene and application
  • A kind of Schistosoma japonicum recombinant protein sjsaplp5 and its coding gene and application
  • A kind of Schistosoma japonicum recombinant protein sjsaplp5 and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Cloning of embodiment 1 Schistosoma japonicum SjSAPLP5 gene

[0046] Primers were designed according to the sequence of the SjSAPLP5 gene (GenBank FN315317.1) (sequence shown in SEQ ID NO.1) and restriction restriction sites were introduced, as follows:

[0047] PF: 5'-CGC GGATCC TCAACATTGAACAGTAATAAT-3' (as shown in SEQ ID NO.3, the italic part represents the protective base of the upstream primer, and the underlined part is the BamH I restriction site of the upstream primer);

[0048] PR: 5'-CTG CTCGAG TGCAAAAGGATATTTCAAA-3' (as shown in SEQ ID NO.4, the italic part represents the protective base of the downstream primer, and the underlined part is the Xho I restriction site of the downstream primer);

[0049] Specific primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0050] Using the cDNA of Schistosoma japonicum 42-day-old male and female adults as a template, perform PCR reaction to amplify the ORF fragment of SjSAPLP5 gene. The reaction sy...

Embodiment 2

[0054] Example 2 Expression and purification of Schistosoma japonicum SjSAPLP5 recombinant protein

[0055] The pET28a(+)-SjSAPLP5 recombinant plasmid with correct sequencing was transformed into expression competent cells Transetta (DE3) (Beijing Quanshijin Biotechnology Co., Ltd.); the positive clones identified by PCR were inoculated into LB liquid medium (containing 50 μg / ml kanamycin) 15mL, cultivate overnight at 37°C, transfer 10mL medium into 1L LB medium (containing 50μg / ml kanamycin) the next day, and continue to culture until OD 600nm When the value is 0.8, IPTG with a final concentration of 1 mM was added to induce expression for 4 hours, the cells were collected by centrifugation, and frozen at -80°C for future use.

[0056] Take a small amount of bacteria before induction and after induction and resuspend in PBS buffer, add SDS-PAGE loading buffer, mix well, boil in boiling water bath for 5min to denature the protein. 10 μl of the pre-induction and post-inductio...

Embodiment 3

[0058] Example 3 Antigenic Detection of Schistosoma japonicum SjSAPLP5 Recombinant Protein

[0059] SDS-PAGE electrophoresis: 100 ng of the SjSAPLP5 recombinant protein obtained in Example 2 was loaded on the sample, and the electrophoresis conditions were: 100V for 20 minutes, 120V for 1 hour.

[0060] Membrane transfer: The protein in the PAGE gel was transferred to the PVDF membrane by wet transfer method, and the electroporation conditions were: ice bath, 100V for 1h.

[0061] Blocking: the PVDF membrane was blocked with 5% skimmed milk powder at room temperature for 2 hours, and washed 3 times with TBST buffer.

[0062] Incubation with primary antibody: BALB / c mouse serum infected with Schistosoma japonicum for 42 days, serum of New Zealand white rabbits infected with Schistosoma japonicum for 42 days, and serum of Schistosoma japonicum patients were added respectively, and mouse anti-His-Tag antibody (Abimate Biomedical Co., Ltd. company) as a positive control, healthy ...

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Abstract

The invention provides a schistosoma japonicum katsurada recombinant protein SjSAPLP5 as well as an encoding gene and an application thereof. The protein provided by the invention has amino acid sequence as shown in SEQ ID No.2 or has amino acid sequence which is formed by replacement, deletion and / or addition of one or more of amino acid residues and has the same function. The invention also provides the encoding gene for encoding the protein. The recombinant protein SjSAPLP5 provided by the invention is excellent in immunogenicity, can be served as an excellent diagnosis antigen, is used for preparing a schistosoma japonicum katsurada diagnostic kit with high sensitivity and high specificity, and is also used for preparing an anti-schistosome vaccine and the drug served as a potential drug effecting target spot and is used for screening and treating the schistosoma japonicum katsurada diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Schistosoma japonicum recombinant protein SjSAPLP5 and its coding gene and application. Background technique [0002] Schistosomiasis is an infectious parasitic disease that seriously threatens human health. It is mainly prevalent in 76 countries and regions in Asia, Africa and Latin America. More than 200 million people are infected worldwide, and nearly 800 million people are threatened by its infection. Schistosomiasis is prevalent in our country. After decades of unremitting efforts, the prevention and control of schistosomiasis in my country has achieved world-renowned achievements. Schistosomiasis has been controlled in endemic areas, but the goal of eventually eliminating schistosomiasis is still a long way to go. Far. [0003] Diagnosis is central to the field of schistosomiasis control. Accurate diagnostic techniques not only have important clinical significance for early...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/11G01N33/68A61K39/00A61P33/12
CPCA61K39/00C07K14/43559G01N33/68G01N2333/43547Y02A50/30
Inventor 陈启军刘帅
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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