Author(s)

He Tian^1#, Shan-E Duan^1#, Xue-Qing Huang², Hao Lu¹, Ji-Yue Liu¹, Hong-Yu Yang¹, Hao Wang¹, Tao-Li Dai^3*

Affiliation(s)

¹Department of Anesthesiology, the First Affiliated Hospital, Jinan University, Guangzhou 510630, P. R. China. 

²Department of Neonatal Surgery, Guangdong women and children's hospital, Guangzhou 511400, P. R. China. 

³Core Laboratory, the First Affiliated Hospital, Jinan University, Guangzhou 510630, P. R. China.

Corresponding Author

Tao-Li Dai

ABSTRACT

Objective: To construct the prokaryotic expressing vectors of cDNA insert fragments for TIA, and recombine GST-fusion proteins.Methods: A phage cDNA library of human aortic endothelial cell line was screened using sera of 19 patients with TIA, the cDNA insert of clones incorporated in pBluescript was cleaved by EcoRI/BamHI/SmalI and XhoI, and the insert fragments were isolated by 1% agarose gel electrophoresis. The expression plasmids of glutathione-S-transferase (GST)-fused proteins were constructed by recombining the cDNA sequence into pGEX-4T-1, 2, 3. The inserted DNA fragments were ligated in frame to pGEX- 4T vectors by using Ligation or In-Fusion protocol. The reaction mixtures were used to transform ECOS? competent E. coli BL-21 and appropriate recombinants were confirmed by DNA sequencing as well as protein expressions through SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The antigenic proteins succeeded in GST-tagged proteins recombinant could be candidate antigens. Results: 21 recombinant pGEX-4T expression vectors containing cDNA sequences of target genes were obtained from 70 SEREX antigens, and the expression of the GST-fusion proteins were shown on SDS-polyacrylamide gels stained with Coomassie staining, and thus, which could be as candidate antigens for further study. Conclusion: Recombination pGEX-4T expression plasmids could be constructed and expressed successfully by Ligation and In-Fusion methods, which may provide basis for the extraction and purification of these antigenic proteins aiming to identify serum antibodies in further study.

KEYWORDS

TIA; SEREX; genetic recombination; prokaryotic expression vector; GST tagged protein

CITE THIS PAPER

He Tian, Shan-E Duan, Xue-Qing Huang, Hao Lu, Ji-Yue Liu, Hong-Yu Yang, Hao Wang, Tao-Li Dai. Construction and expression of prokaryotic expression vector of TIA associated antigens by SEREX screening. Acta Translational Medicine. 2020, 3(1): 41-51.

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