Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Schistosoma japonicum chymotrypsin-like protease (SjCTRL) as well as preparation method and application thereof

A recombinant antigen protein, recombinant protein technology, applied in cell biology research, molecular field, can solve the problem that there is no public report against schistosoma japonicum vaccine and so on

Active Publication Date: 2015-12-16
FUDAN UNIV +1
View PDF5 Cites 1 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Schistosoma japonicum chymotrypsin-like protease (SjCTRL) as well as preparation method and application thereof
  • Schistosoma japonicum chymotrypsin-like protease (SjCTRL) as well as preparation method and application thereof
  • Schistosoma japonicum chymotrypsin-like protease (SjCTRL) as well as preparation method and application 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 (GenbankFN317561.1) (sequence shown in SEQ ID NO.1), use the PrimerPremier5.0 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 BamHI restriction site of the upstream primer and the XhoI 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] Construction of embodiment 2 SjCTRL gene recombinant plasmid pET-28a(+)-SjCTRL

[0070] 2.1 PCR product double digestion and recovery

[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 culture it overnight at 37° C. in 5 ml LB medium (containing 50 μg / ml kanamycin). 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 remaining cul...

Embodiment 3

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

[0085] 3.1 Transformation of ligated products into E.coliDH5α 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 dete...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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) antigen 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, Africa and In Latin America, 600 million people are threatened and more than 200 million people are 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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com