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| Cloning, expression, purification of human blood-brain barrier permeability related gene Caveolin-1 |
| ZHAO Shutao XU Yongjian CHEN Haoyun |
| Department of Intensive Care, Jinshazhou Hospital of Guangzhou University of Chinese Medicine, Guangdong Province, Guangzhou 510168, China |
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Abstract Objective To clone Caveolin-1, a gene related to human blood-brain barrier permeability, and express it in Escherichia coli. and to purify and identify the protein finally. Methods According to the sequence of Caveolin-1 gene contained in the gene bank, the upstream and downstream primers were designed. The Caveolin-1 double strand was synthesized by PCR and then recombined with pET28a (+) vector. Positive clones were screened and identified by DNA sequence analysis. The constructed recombinant plasmid pET28a-Caveolin-1 was transfected into E. coli BL21 (DE3), and the expression of the fusion protein was induced by IPTG. The expressed product was purified by Ni2+-NTA affinity chromatography, identified by SDS-PAGE and Western blot. Results The Caveolin-1 DNA fragment amplified by PCR was identified as human Caveolin-1 gene. The recombinant human Caveolin-1 fusion protein was expressed by IPTG-induced DE3 strain containing the recombinant plasmid pET28a-Caveolin-1. The recombinant protein was purified by Ni2+-NTA affinity chromatography to obtain a fusion protein of higher purity. Conclusion This study successfully cloned the blood-brain barrier permeability-related gene Caveolin-1, and expressed and purified the protein to obtain a higher purity fusion protein, which laid a foundation for further relevant clinical research.
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