Schistosoma japonicum sjctrl recombinant antigen protein and preparation method and use thereof

A technology for recombining antigenic proteins and recombinant proteins, which is applied in the field of molecular and cell biology research, and can solve problems such as the absence of public reports of anti-Schistosomiasis vaccines

Active Publication Date: 2019-03-15
FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Before the present invention, there was no public report related to the recombinant antigen protein of Schistosoma japonicum SjCTRL of the present invention and its use in the preparation of anti-Schistosoma japonicum vaccine

Method used

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  • Schistosoma japonicum sjctrl recombinant antigen protein and preparation method and use thereof
  • Schistosoma japonicum sjctrl recombinant antigen protein and preparation method and use thereof
  • Schistosoma japonicum sjctrl recombinant antigen protein and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Cloning of Schistosoma japonicum SjCTRL gene

[0048] 1.1 Amplification and purification of SjCTRL gene fragments:

[0049] 1.1.1 According to the sequence of the SjCTRL gene (Genbank FN317561.1) (sequence shown in SEQ ID NO.1), use Primer Premier 5.0 software to design a pair of specific primers, as follows:

[0050] PF:5′- CG AATTTAGAATATCGTATACAAAATGGTT-3' (as shown in SEQ ID NO.3);

[0051] PR:5′- CC TCAACCACTGCCTGCTATTG-3' (as shown in SEQ ID NO.4);

[0052] The single underline is the protective base of the upstream and downstream primers, the double underline is the BamH I restriction site of the upstream primer and the Xho I restriction site of the downstream primer. Specific primers were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.

[0053] 1.1.2 Using the cDNA of Schistosoma japonicum as a template, carry out PCR reaction to amplify the SjCTRL gene, the reaction system is as follows:

[0054]

[0055] Reaction conditions: ...

Embodiment 2

[0069] Example 2 Construction of SjCTRL Gene Recombination Plasmid pET-28a(+)-SjCTRL

[0070] 2.1 Double digestion and recovery of PCR products

[0071] The recovered PCR target fragment was double-digested overnight in a water bath at 37°C. The enzyme digestion reaction system was as follows:

[0072]

[0073] Digested products were subjected to 1.2% agarose gel (GoldView TM Nucleic acid dye) electrophoresis, cut off the target band, and recover the DNA molecule again, the recovery method is the same as 1.1.3 in Example 1, and the recovered product is stored at -20°C.

[0074] 2.2 Preparation of pET-28a(+) Empty Plasmid (AxyPrep Plasmid Miniprep Kit)

[0075]Pick a single colony containing the pET-28a(+) plasmid on an LB plate (containing 50 μg / ml kanamycin) and place it in 5 ml LB medium (containing 50 μg / ml kanamycin), and culture overnight at 37°C . On the next day, transfer 1ml of the culture solution into a 1.5ml centrifuge tube and store at 4°C as a strain. The ...

Embodiment 3

[0084] Example 3 Identification of Schistosoma japonicum SjCTRL Expression Vector

[0085] 3.1 Transformation of Ligation Products into E.coli DH5α Competent Cells

[0086] Gently mix the ligation product in 2.4 of Example 2 with E.coli DH5α competent cells, ice-bath for 30 minutes, heat shock in a water bath at 42°C for 1.5 minutes, and ice-bath again for 5 minutes. Add 900 μl of SOC medium to the culture tube under sterile conditions, shake at 37° C. and 200 rpm for 1 hour. Centrifuge the cultured bacterial solution at 3500 rpm for 3 minutes, discard most of the supernatant, and leave about 100 μl of medium to resuspend the bacterial cells. The resuspension was evenly spread on LB plates (containing 50 μg / ml kanamycin), cultured upside down at 37° C. overnight, and the growth of colonies was observed.

[0087] 3.2 PCR identification of recombinant plasmids

[0088] A single colony on the plate was randomly selected for colony PCR identification, and the PCR product was de...

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Abstract

The invention discloses a Schistosoma japonicum chymotrypsin-like protease (SjCTRL), a protein with an amino acid sequence shown in the SEQ ID NO.2, or a protein which has the same function and is formed through replacement, deletion or insertion of one or more amino acids for the protein. Besides, the invention further discloses a preparation method of the SjCTRL. The preparation method comprises steps such as transmembrane structure removed gene sequence amplification of the SjCTRL, recombinant plasmid construction and identification, induction expression and purification of the SjCTRL and the like, and the immunoprotection of the SjCTRL in anti-schistosoma-infection of mice is evaluated. It is preliminarily verified that the SjCTRL can reduce the number of adults of C57BL / 6 mice infected by Schistosoma japonicum and the number of eggs in livers to a certain extent and can serve as a target of a potential anti-schistosoma vaccine.

Description

technical field [0001] The invention relates to the field of molecular and cell biology research, in particular to a recombinant Schistosoma japonicum chymotrypsin-like protease (SjCTRL) protein and a preparation method thereof. In addition, the present invention also relates to the use of the Schistosoma japonicum SjCTRL recombinant antigen protein. Background technique [0002] Schistosomiasis is a zoonotic parasitic disease (parasitic zoonoses), which is the second largest parasitic disease after malaria in the world. It is prevalent in 76 countries and regions around the world, mainly distributed in Asia and Africa. and Latin America, with 600 million people threatened and more than 200 million people infected. China was once one of the countries seriously affected by Schistosoma japonicum. It was once prevalent in 12 provinces south of the Yangtze River, with more than 12 million patients. Only Schistosoma japonicum is endemic in my country, and it is mainly distribut...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/11C12N15/70A61K39/00A61P33/12
CPCA61K39/0003C07K14/43559Y02A50/30
Inventor 胡薇柴日奕王吉鹏徐斌李健张瑞祥刘牧辛玥莫筱瑾张颋
Owner FUDAN UNIV
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