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Schistosoma japonicum recombinant antigen, and preparation method and application thereof

A technology of recombinant antigen and schistosomiasis, applied in the field of bioengineering, can solve the problem of no research reports and other problems, and achieve the effect of good application value

Inactive Publication Date: 2015-12-09
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

[0006] But so far, there is no research report on the application of SjTOR recombinant protein of Schistosoma japonicum as a vaccine against Schistosoma japonicum

Method used

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  • Schistosoma japonicum recombinant antigen, and preparation method and application thereof
  • Schistosoma japonicum recombinant antigen, and preparation method and application thereof
  • Schistosoma japonicum recombinant antigen, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Expression and purification of Schistosoma japonicum recombinant antigen

[0040] 1. Method

[0041] 1.1 Construction of recombinant expression plasmid

[0042] According to the gene sequence of SjTOR (AY814912.1) included in NCBI, primers were designed, upstream primer: 5'-GCGGAATTCTCGTTTCTTTTCCTA-3' (SEQ ID NO. 3); downstream primer: 5'-CGCCTCGAGGAAAGTTGTCACACAGACAGC-3' (SEQ ID NO. 4), with To amplify the SjTOR gene fragment. Using the cDNA of the 42-day worm of Schistosoma japonicum as a template, PCR amplifies the cDNA fragment containing ORF, and the reaction composition is as follows:

[0043]

[0044] PCR reaction conditions: 94°C for 5min, 94°C denaturation for 30sec, 55°C annealing for 30sec, 72°C extension for 90sec, a total of 30 cycles; finally 72°C for 15min. After the PCR product is purified, it is connected to the pMD19-T vector, transformed into DH5α cells, a single clone is picked, and the PCR bacteria liquid is identified. The positive clone is sen...

Embodiment 2

[0063] Example 2 Antigenicity detection of recombinant protein rSjTOR-ed1 of Schistosoma japonicum

[0064] 1. WesternBlotting analysis of recombinant protein antigenicity

[0065] The recombinant protein rSjTOR-ed1 purified by the Ni column was subjected to SDS-PAGE electrophoresis, and then transferred to the NC membrane with 130mA for 1h at 4℃, and the antiserum against the recombinant protein was used as the first antibody to analyze the antigen of the recombinant protein Sex.

[0066] 2. WesternBlot analysis of the results of recombinant protein antigenicity

[0067] WesternBlot analysis results show that the recombinant protein rSjTOR-ed1 can be recognized by the recombinant protein immune serum and positive serum respectively, indicating that the recombinant protein rSjTOR-ed1 has good immunogenicity ( Figure 5 , 6 ). in Figure 5 Middle, M: Marker; A: Primary antibody is mouse positive serum; B: Primary antibody is normal mouse serum. in Image 6 Middle, M: Marker; A: Prima...

Embodiment 3

[0068] Example 3 Immune prevention experiment of Schistosoma japonicum recombinant protein rSjTOR-ed1

[0069] 1. Method steps

[0070] 1.1 Animal immune protection experiment

[0071] The 6-week-old BALB / c mice were divided into three groups, namely the recombinant protein SjTOR-ed1 immunization group, the 206 adjuvant control group and the PBS control group, with 10 mice in each group. Each time mice in the recombinant protein immunized group were immunized, each mouse was injected subcutaneously with 100 μL of recombinant protein SjTOR-ed1 (20 μg) and 206 adjuvant emulsion. In the 206 adjuvant control group, each mouse was injected subcutaneously with 100 μL of 206 adjuvant and PBS emulsion. In the PBS control group, each mouse was injected subcutaneously with 100 μL PBS each time. A total of three immunizations were carried out with an interval of 2 weeks each time. 7 days after each immunization, blood was collected from the orbit of each mouse, serum was collected, and store...

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Abstract

The invention discloses a Schistosoma japonicum recombinant antigen. The recombinant antigen is prepared through expressing a recombinant vector containing first extramembrane fragment gene at the N-end of a Schistosoma japonicum trispanning orphan receptor. The invention also discloses a preparation method of the Schistosoma japonicum recombinant antigen, and an application of the Schistosoma japonicum recombinant antigen in preparation of schistosomiasis prevention or treatment vaccines or drugs. The Schistosoma japonicum recombinant antigen in mouse immunization experiments can induce generation of anti-recombinant antigen specific IgG, IgG1 and IgG2a antibodies and allows the antibodies to reach a high level, and the Schistosoma japonicum recombinant antigen in animal protection experiments induces the worm reduction rate of 32.4% and the ovum reduction rate of 41.41%, so the recombinant antigen is suitable for being used as an anti-schistosomiasis candidate vaccine, and has very good application prospect.

Description

Technical field [0001] The invention relates to the technical field of bioengineering, in particular to a Schistosoma japonicum recombinant antigen and its preparation method and application. Background technique [0002] Schistosomiasis japonica is a zoonotic parasitic disease that is endemic in southern my country that seriously harms human and animal health, and affects social and economic development. It is one of the most important public health problems in my country. Since the 1950s, my country has made great achievements in the prevention and control of schistosomiasis. However, because the ecological environment of schistosomiasis epidemic in some traditional epidemic areas (Dongting Lake area, Poyang Lake area and some rivers and lakes) is difficult to effectively change, it is necessary to stop schistosomiasis Communication is still a long-term, arduous and complex task. At present, the main control method for end-host hosts (human, cattle, sheep, etc.) is drug treatme...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C07K14/435C07K16/18A61K39/00A61K38/17A61P33/12
CPCY02A50/30
Inventor 傅志强马帅洪炀林矫矫伍妙梨陆珂李浩朱传刚韩艳辉贾秉光曹晓丹韩倩宰金丽
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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