A study to construct three prokaryotic expression vectors and its expression in adenosine deaminase
10.3969/j.issn.1673-4130.2019.03.007
- VernacularTitle:腺苷脱氨酶三种原核表达载体的构建及蛋白表达
- Author:
Liqiao HAN
1
;
Lu ZHANG
;
Haibiao LIN
;
Xiaoting HUANG
;
Xinzhong WU
;
Xianzhang HUANG
;
Junhua ZHUANG
Author Information
1. 广州中医药大学第二附属医院
- Keywords:
adenosine deaminase;
ADA;
prokaryotic vector;
pET-28b;
pET-32a;
pHSIE;
protein expression
- From:
International Journal of Laboratory Medicine
2019;40(3):281-284,289
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express adenosine deaminase protein by molecular cloning technique.MethodsTotal RNA was extracted from human leukocytes and the cDNA was obtained by reverse transcription.Whereupon the cDNA was used as a template to amplify adenosine deaminase gene by polymerase chain reaction (PCR) and then integrated it into three prokaryotic plasmids pET-28 b, pET-32 a (+) and pHSIE.The plasmid with the correct sequencing was transformed into E.coli BL21 (DE3) by CaCl2 method for the protein expression.The expression activity of these fusion proteins were detected by Western-blot and SDS-PAGE, with the optimized expression conditions.Results Complete fusion of target gene and three prokaryotic plasmids was observed through sequencing.The expressed and accurate ADA protein was identified by Westernblot and SDS-PAGE.The optimal expression conditions were observed:the protein expression would be induced with 0.4 mmol/L IPTG and incubated at 16℃for 24 hours.Conclusion The prokaryotic vectors of adenosine deaminase (BL21+pET-28 b+ADA, BL21+pET-32 a+ADA, BL21+pHSIE+ADA) were successfully constructed and efficiently expressed.
