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| Prokaryotic Expression, Antibody Preparation of GP2 in Bactrian Camel (Camelus bactrianus) and Its Expression in Ileum |
| LIU Ke-Jiang, LI Pei-Xuan, ZHANG Rui, WANG Ting-Wei, SUO Nan-Ji, WANG Wen-Hui* |
| College of Animal Medicine, Gansu Agricultural University, Lanzhou 730070, China |
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Abstract Glycoprotein 2 (GP2) is a kind of glycosylphosphatidylinositol-anchored proteins (GPI-APs) produced by mucous glands and secretory cells, it can ingest pathogenic bacteria in the intestinal cavity and is of great significance to maintain the homeostasis of the body. In order to obtain the antibody against Bactrian camel (Camelus bactrianus) GP2, the bactrian camel GP2 gene was cloned into pET-28a, constructing the recombinant plasmid pET-28a-GP2, the recombinant plasmid pET-28a-GP2 was then transferred into Escherichia coli BL21 for induction and expression. After the optimization of induction conditions, the expression of GP2 recombinant protein was detected by SDS-PAGE and Western blot. To prepare rabbit polyclonal antibodies against GP2 with target protein, The titer and specificity of the antibody was detected by ELISA and Western blot, immunohistochemical staining was used to understand its expression in ileum. The results showed that the size of GP2 protein was about 65 kD, the optimal induction conditions were 0.5 mmol/L isopropyl-beta-D-thiogalactopyranoside (IPTG) 6 h, mainly expressed in the form of inclusion bodies. The polyclonal antibody was used at a titer of 1∶2.56×105 to specifically recognize recombinant protein GP2 as determined by ELISA and Western blot. Immunohistochemical staining results showed that GP2 is mainly expressed in follicular associated epithelium of ileum, and expressed on the cell membrane of microfold cell (M cell). This study provides a reference for the follow-up study of GP2, enriches the relevant theories of mucosal immunity, solves the problem of no commercially available antibodies on the market at present, and provides antibody support for the development of new drugs.
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Received: 08 August 2022
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Corresponding Authors:
* wwh777@126.com
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