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Genes highly expressed in Schistosoma japonicum schistosomulum and encoded proteins and application thereof

A schistosomiasis, high-expression technology, which is applied to highly-expressed genes and their encoded proteins and application fields, can solve the problems of low specificity in schistosomiasis diagnosis, unsuitable standardization of reagents, complex components, etc., and achieves stable results, high reproducibility, and high reproducibility. Sensitive effect

Active Publication Date: 2019-10-15
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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  • Genes highly expressed in Schistosoma japonicum schistosomulum and encoded proteins and application thereof
  • Genes highly expressed in Schistosoma japonicum schistosomulum and encoded proteins and application thereof
  • Genes highly expressed in Schistosoma japonicum schistosomulum and encoded proteins and application thereof

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, SjScP88 published in GenBank (the putative transcriptome data of S. japonicum BU802382, FN315317.1, FN357371.1, FN357552.1), primers were designed and introduced restriction sites and primer sequences 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 TTCCAGGAAGTTAAATTATGCCGT-3’,

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

[0080] SjScP88:

[0081] PF: 5’- GGGGACAAGTTTGTACAAAAAAGCAGGCTT GGTAATGGAAAACGAAATTTCAAC-3’,

[0082] PR: 5’- GGGGAC...

Embodiment 2

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

[0089] Inoculate 15 mL of LB liquid medium (containing 50μg / ml ampicillin) into the clones identified by the above PCR as being positive, and incubate overnight at 37°C at 200 rpm. The next day, transfer 10 mL of medium to LB medium (containing 50μg / ml ampicillin). Penicillin) 1L, continue to cultivate to OD 600nm The value was 0.8, and IPTG with a final concentration of 1 mM was added for induction. The cells were expressed at 18°C ​​at 140 rpm for 16 hours, and the cells were collected by centrifugation and stored at -80°C for later use.

[0090] Take a small amount of pre-induction and post-induction bacteria and suspend them in PBS buffer, add SDS-PAGE loading buffer, mix well and cook for 5 minutes in a boiling water bath to denature the protein.

[0091] Add 10 μl of the sample before and after induction to each sample well, and perform SDS-PAGE an...

Embodiment 3

[0100] Example 3 Antigenicity detection of recombinant proteins of Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88

[0101] SDS-PAGE electrophoresis: take 100ng of recombinant protein and load the sample, the electrophoresis conditions are: 100V 20min, 120V 1h.

[0102] Transfer membrane: Use the wet transfer method to transfer the protein in the PAGE gel to the PVDF membrane. The electrotransfer conditions are: ice bath, 100V1h.

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

[0104] Incubation with primary antibody: add the serum of BALB / c mice infected with Schistosoma japonicum for 42 days, the serum of New Zealand white rabbits infected with Schistosoma japonicum for 42 days, and the serum of patients with Schistosoma japonicum, with mouse anti-His-tag antibody (Abimart Company) as a positive control, healthy mouse serum as a negative control (diluted with blocking solution 1:500)...

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Abstract

The present invention provides genes highly expressed in Schistosoma japonicum schistosomulum and encoded proteins and application thereof. A whole-genome expression profile chip of Schistosoma japonicum is utilized to screen a series of genes highly expressed in Schistosoma japonicum schistosomulum, and antigen proteins encoded by genes which are SjScP27, SjScP80, SjScP84 and SjScP88 can be specifically recognized by serum of patients with schistosomiasis and show obvious positive reactions. ELISA detection proves that the antigen proteins have high sensitivity and specificity for Schistosomajaponicum detection, and can be used for developing diagnostic reagents for schistosomiasis japonica.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a gene highly expressed in Schistosoma japonicum juvenile worms and its coded proteins and applications. 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 japonicum is prevalent in my country. After decades of unremitting efforts, my country's schistosomiasis prevention and control work has achieved world-renowned achievements. Schistosomiasis has been controlled in endemic areas, but there is still a heavy task to achieve the ultimate goal of eliminating schistosomiasis far. [0003] Diagnosis is the central link in the field of schistosomiasis control. Accurate diagnostic technology not only ha...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12G01N33/68A61K39/00A61P33/12
CPCC07K14/43559G01N33/6893A61K39/0003A61P33/12G01N2333/43547Y02A50/30
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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