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Schistosoma japonicum katsurada recombinant protein SjSAPLP4 as well as encoding gene and application thereof

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

Active Publication Date: 2016-03-09
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

Before the present invention, there was no published report related to the recombinant antigen protein of Schistosoma japonicum SjSAPLP4 of the present invention and its use in the diagnosis of Schistosomiasis japonicum

Method used

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  • Schistosoma japonicum katsurada recombinant protein SjSAPLP4 as well as encoding gene and application thereof
  • Schistosoma japonicum katsurada recombinant protein SjSAPLP4 as well as encoding gene and application thereof
  • Schistosoma japonicum katsurada recombinant protein SjSAPLP4 as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Cloning of embodiment 1 Schistosoma japonicum SjSAPLP4 gene

[0046] According to the sequence of the SjSAPLP4 gene (GenBankFN315317.1) (sequence shown in SEQ ID NO.1), primers were designed and restriction restriction sites were introduced, as follows:

[0047] PF: 5'- GGATCC AACCACACTGAGTTGGACAT-3' (as shown in SEQIDNO.3, the italic part represents the protective base of the upstream primer, and the underlined part is the BamHI restriction site of the upstream primer);

[0048] PR: 5'- CTCGAG TGGGCATAACTGTATTGTCT-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 XhoI restriction site of the downstream primer);

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

[0050] Using the cDNA of 42-day-old male and female adults of Schistosoma japonicum as a template, perform PCR reaction to amplify the ORF fragment of SjSAPLP4 gene. The reaction syste...

Embodiment 2

[0054] Example 2 Expression and purification of Schistosoma japonicum SjSAPLP4 recombinant protein

[0055] The pET28a(+)-SjSAPLP4 recombinant plasmid with correct sequencing was transformed into expression competent cells Transetta (DE3) (Beijing Quanshijin Biotechnology Co., Ltd.); the clones identified as positive 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-induc...

Embodiment 3

[0058] Example 3 Antigenic Detection of Schistosoma japonicum SjSAPLP4 Recombinant Protein

[0059] SDS-PAGE electrophoresis: 100 ng of the SjSAPLP4 recombinant protein obtained in Example 2 was loaded on the sample, and the electrophoresis conditions were: 100V20min, 120V1h.

[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, 100V1h.

[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 mouse serum as a negativ...

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Abstract

The invention provides a schistosoma japonicum katsurada recombinant protein SjSAPLP4 as well as encoding gene and application thereof. The protein has an amino acid sequence shown as SEQ ID NO.2 or an amino acid sequence having a same function, formed by replacing, omitting and / or adding one or more amino acid residues for the amino acid sequence shown as SEQ ID No.2. The invention also provides a gene sequence for encoding the protein. The recombinant protein SjSAPLP4 is good in immunogenicity, can be used as an excellent diagnostic antigen, can be used for preparing a schistosoma japonicum katsurada diagnosis kit having high sensitivity and high specificity, also can be used for preparing an anti-schistosome vaccine and can be used as a potential drug acting target spot to screen the drug for treating the schistosoma japonicum katsurada.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Schistosoma japonicum recombinant protein SjSAPLP4 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 Applications(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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