绿脓杆菌外毒素PE38KDEL重组蛋白原核表达及多克隆抗体的制备

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Abstract Objective: To prepare PE38KDEL recombinant protein from Pseudomonas exotoxin A in prokaryotic expression system and prepare the PE38KDEL specific polyclonal antibody. Methods: The C-terminal 609-613 amine acid REDLK from Pseudomonas aeruginosa exotoxin (PE) genes (PE38) was mutanted to KDEL. After prokaryotic codon optimization (http://www.jcat.de), the full gene sequences were synthesized and then cloned into expression vector pET32a (+) prokaryotic expression vector inserted into Hind III and Xho I and constructed the recombinant plasmid pET32a (+)/PE38KDEL. After sequenced, the plasmid was transformed into E.coli BL21 (DE3) and induced by isopropyl beta-D-1-thiogalactoside (IPTG) to get the PE38KDEL protein. The recombinant protein was purified by Ni-NTA affinity chromatography, SDS-PAGE and Western blot were used to identify it. Then, the rabbits were immunized by the PE38KDEL purified protein. The sera were collected before and after immunized. The antibody reaction and titer were detected by ELISA. Results: The pET32a (+)/PE38KDEL recombinant plasmid was successfully constructed and the protein was expressed and purified in the prokaryotic expression system. The protein molecular weight was about 57 kDa which was in accordance with the expected theoretical value. The PE38KDEL polyclonal antibody was obtained by immunization of rabbits and the titer of it was as high as 1:60 000. Conclusion: The PE38KDEL recombinant protein can induce the rabbit to produce serum polyclonal antibody which has high titer and strong specificity and can be used for further study which laid a solid foundation on the biology and immunology function.

Key words： Pseudomonas aeruginosa exotoxins PE38KDEL polyclonal antibody rabbits

Received: 13 May 2016

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	CEN Danwei
	SONG Yiling
	ZHANG Chanqiong
	MAO Shanshan
	YE Xiaoxian
	CHEN Jun
	CHEN Shao
	ZHU Shanli
	ZHANG Lifang.?

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https://xb.wmu.edu.cn/EN/ OR https://xb.wmu.edu.cn/EN/Y2017/V47/I1/1

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