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Highly expressed genes and their encoded proteins in Schistosoma japonicum larvae and their applications

A high-expression technology for schistosomiasis, applied to high-expression genes and their encoded proteins and application fields, can solve the problems of unsuitable standardization of reagents, low diagnostic specificity of schistosomiasis, complex components, etc.

Active Publication Date: 2020-10-30
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

Currently, the most commonly used antigens for the immunodiagnosis of schistosomiasis are adult worm antigen (AWA) and egg antigen (Soluble egg antigen, SEA) extracted from schistosomiasis. The composition of the antigen is complex (consisting of thousands of schistosomiasis proteins), and there is a serious cross-reaction with other parasite-infected sera. The specificity of schistosomiasis diagnosis is not high, and the reagents produced should not be standardized.

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  • Highly expressed genes and their encoded proteins in Schistosoma japonicum larvae and their applications
  • Highly expressed genes and their encoded proteins in Schistosoma japonicum larvae and their applications
  • Highly expressed genes and their encoded proteins in Schistosoma japonicum larvae and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Cloning of Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 genes

[0070] According to the sequences of SjScP27, SjScP80, SjScP84, and SjScP88 genes (the putative transcriptome data of S. japonicum BU802382, FN315317.1, FN357371.1, FN357552.1) published in GenBank, primers were designed and restriction sites were introduced. as follows:

[0071] SjScP27:

[0072] PF: 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTTC AATCTCATAGTATCAGATACAAC-3',

[0073] PR: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCCTA TCACTTACCATCAAGATGAAAT-3'

[0074] SjScP80:

[0075] PF: 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTT AAAGCCTTTCGTCGGTGATGC-3',

[0076] PR: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCCTA GTGAAACCCAACTGGACATA-3'

[0077] SjScP84:

[0078] PF: 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTTC TTCCAGGAAGTTAAAATTATGCCGT-3',

[0079] PR: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCCTA TCAACTCACCTCACCAACATAA-3'

[0080] SjScP88:

[0081] PF: 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTT GGTAATGGAAAACGAAATTTCAAC...

Embodiment 2

[0088] Example 2 Expression and purification of Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 recombinant protein

[0089] The clones identified as positive by the above PCR were inoculated into 15 mL of LB liquid medium (containing 50 μg / ml ampicillin). Penicillin) 1L, continue to culture to OD 600nm The value was 0.8, and then IPTG with a final concentration of 1 mM was added for induction, expressed at 140 rpm at 18°C ​​for 16 hours, collected by centrifugation, and frozen at -80°C for future use.

[0090] 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.

[0091] 10 μl of the pre-induction and post-induction samples were added to each loading well for SDS-PAGE analysis (5% for stacking gel, 12% for separating gel).

[0092] The pDEST17-SjScP27, pDEST17-SjScP80, pDEST17-SjScP84, pDEST17-SjScP88 recombinant plasmids ...

Embodiment 3

[0100] Example 3 Antigenic Detection of Schistosoma japonicum SjScP27, SjScP80, SjScP84, and SjScP88 Recombinant Proteins

[0101] SDS-PAGE electrophoresis: 100ng of recombinant protein was loaded on the sample, and the electrophoresis conditions were: 100V for 20min, 120V for 1h.

[0102] 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.

[0103] Blocking: the PVDF membrane was blocked with 5% skimmed milk powder at room temperature for 2 hours, and washed 3 times with TBST buffer.

[0104] 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 patients with Schistosoma japonicum were added respectively, and mouse anti-His-tag antibody (Abimate Biomedical Co., Ltd. company) as a positive control, healthy mouse ser...

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Abstract

The invention provides a gene highly expressed in Schistosoma japonicum larvae, its encoded protein and its application. The present invention screens a series of highly expressed genes in Schistosoma japonicum larvae by using the whole genome expression spectrum chip of Schistosoma japonicum, wherein the antigenic proteins encoded by the genes SjScP27, SjScP80, SjScP84, and SjScP88 can be specifically recognized by the serum of schistosomiasis patients, and present strong positive reaction. ELISA detection showed that these antigenic proteins had high sensitivity and specificity in detecting schistosomiasis, and could be used in the development of diagnostic reagents for schistosomiasis japonicum.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a gene highly expressed in Schistosoma japonicum larvae, its encoded protein and its 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 si...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/435C12Q1/6888G01N33/68A61P33/12
CPCC07K14/43559C12Q1/6888G01N33/68G01N2333/43547
Inventor 侯楠陈启军刘帅朴贤玉
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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