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Schistosoma japonicum calcium-binding EF-hand domain containing protein (SjEFCAB) recombinant antigen protein and preparation method and application thereof

A technology of recombinant antigenic protein and recombinant protein, which is applied in cell biology research and molecular fields, can solve the problem that there is no public report of Schistosoma japonicum SjEFCAB recombinant antigenic protein, and achieve high sensitivity and specificity

Inactive Publication Date: 2013-10-30
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Before the present invention, there was no public report related to the recombinant antigen protein of Schistosoma japonicum SjEFCAB of the present invention and its use in the diagnosis of Schistosomiasis japonicum

Method used

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  • Schistosoma japonicum calcium-binding EF-hand domain containing protein (SjEFCAB) recombinant antigen protein and preparation method and application thereof
  • Schistosoma japonicum calcium-binding EF-hand domain containing protein (SjEFCAB) recombinant antigen protein and preparation method and application thereof
  • Schistosoma japonicum calcium-binding EF-hand domain containing protein (SjEFCAB) recombinant antigen protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Cloning of Schistosoma japonicum SjEFCAB Gene

[0049] 1.1 Amplification and purification of SjEFCAB gene fragments:

[0050] 11.1 According to the sequence of the SjEFCAB gene (Genbank accession number FN324043.1) (sequence shown in SEQ ID NO.1), use PrimerPremier5.0 software to design a pair of specific primers, as follows:

[0051] PF: 5'-CCG GAATTC ATGAAACCATCAATGGAAGAGA-3' (as shown in SEQ ID NO.3);

[0052] PR: 5'-CCG CTCGAG TTAACAAGAGAACATTTTAACC-3' (as shown in SEQ ID NO.4);

[0053] The parts in italics are the protective bases of the upstream and downstream primers, and the bold parts are the EcoR I restriction sites of the upstream primers and the Xho I restriction sites of the downstream primers. The above-mentioned specific primers were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd. 11.2 Using the pBluescript-SjEFCAB plasmid as a template, perform a PCR reaction to amplify the SjEFCAB gene. The reaction system is as follows:

[0054...

Embodiment 2

[0069] Example 2 Construction of SjEFCAB Gene Recombination Plasmid pGEX-4T-1-SjEFCAB

[0070] 2.1 Double enzyme 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 1.5% 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 11.3 in Example 1, and the recovered product is stored at -20°C.

[0074] 2.2 Preparation of pGEX-4T-1 Empty Plasmid (AxyPrep Plasmid Miniprep Kit)

[0075]Pick a single colony containing the pGEX-4T-1 plasmid on an LB plate (containing 100 μg / ml ampicillin) and culture it overnight at 37° C. in 5 ml 2×YTA medium (containing 100 μg / ml ampicillin). 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 SjEFCAB 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. Spread the resuspension evenly on LB plates (containing 100 μg / ml of ampicillin), incubate overnight at 37° C., and observe the growth of colonies.

[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 detected by agaro...

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Abstract

The invention discloses a preparation method of a Schistosoma japonicum calcium-binding EF-hand domain containing protein (SjEFCAB) recombinant antigen protein. The preparation method comprises the following steps of: designing SEQ ID NO.3 or a complementary chain 5' thereof as a primer and SEQ ID NO.4 or a complementary chain 3' thereof as a primer, and carrying out amplification on a Schistosoma japonicum SjEFCAB gene sequence; establishing and identifying Schistosoma japonicum SjEFCAB recombinant plasmids; and carrying out induction expression, purification and the like on recombinant protein. Besides, the invention further discloses an application of the Schistosoma japonicum SjEFCAB recombinant antigen protein prepared by using the method in preparation of products for detecting a serum antibody of a Schistosoma japonicum patient. Enzyme linked immunosorbent assay (ELISA) proves that the Schistosoma japonicum SjEFCAB recombinant antigen protein has higher sensitivity and specificity when used for diagnosing the Schistosoma japonicum, is a potential candidate target of diagnosis, and can be used as a target antigen for diagnosing the Schistosoma japonicum.

Description

technical field [0001] The invention relates to the field of molecular and cell biology research, in particular to a recombinant antigenic protein of Schistosoma japonicum containing EF-hand domain calcium-binding protein (SjEFCAB) and a preparation method thereof. In addition, the present invention also relates to the application of the SjEFCAB recombinant antigen protein of Schistosoma japonicum in the preparation of products for detecting serum antibodies of patients with Schistosoma japonicum. Background technique [0002] Schistosomiasis is a zoonotic parasitic disease that seriously endangers human health and hinders social and economic development in endemic areas. There are three main types of schistosomiasis that parasitize humans and animals, namely Schistosoma mansoni, Schistosoma japonicum and Schistosoma haematobium. In my country, only schistosomiasis is prevalent, and it is one of the five major parasitic diseases planned by the Ministry of Health. By 2009, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/70C07K14/435G01N33/68
Inventor 胡薇卢艳徐斌莫筱瑾鞠川陈绅波冯正
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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