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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摘要目的：通过原核表达系统制备绿脓杆菌外毒素（PE）PE38KDEL重组蛋白，并制备特异性兔免疫血清多克隆抗体。方法：选择PE部分基因（PE38），并将C端609-613位的氨基酸REDLK突变为KDEL，通过原核密码子优化（http://www.jcat.de）后全基因合成，经Hind III和Xho I酶切位点克隆至pET32a（+）原核表达载体，构建pET32a（+）/PE38KDEL重组质粒，将测序正确的重组质粒转化到E.coli BL21（DE3）感受态细菌中，经异丙基硫代半乳糖苷（IPTG）诱导表达重组PE38KDEL蛋白，经Ni-NTA亲和层析法制备纯化蛋白，采用SDS-PAGE电泳和Western blot法进行鉴定。进一步将PE38KDEL纯化蛋白免疫日本大耳白兔制备PE38KDEL特异性多克隆抗体，并采集免疫前后血清，用ELISA法检测免疫后的抗体反应和效价。结果：成功构建了pET32a（+）/PE38KDEL重组质粒。在原核表达系统中该质粒成功表达并获得纯化的PE38KDEL蛋白。SDS-PAGE电泳显示蛋白分子质量约为57 kDa，与预计理论值大小相符合。Western blot法检测结果显示，在分子质量约57 kDa处出现单一条带。通过免疫大耳白兔成功获得PE38KDEL特异性多克隆抗体，抗体效价高达1:60 000。结论：PE38KDEL蛋白可诱导兔产生特异性多克隆血清抗体，且该抗体效价高、特异性强，为进一步研究基于PE38KDEL毒素的生物学和免疫学等功能奠定了基础。

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	岑丹维
	宋易玲
	张婵琼
	毛珊珊
	叶晓鲜
	陈俊
	陈韶
	朱珊丽
	张丽芳

关键词 ：铜绿假单胞菌, 外毒素类, PE38KDEL, 多克隆抗体, 兔

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

收稿日期: 2016-05-13

基金资助:国家自然科学基金资助项目（81372447）。

通讯作者: 张丽芳，教授，博士生导师，Email：wenzhouzlf@126.com。

作者简介: 岑丹维（1991-），女，浙江舟山人，硕士生。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2017/V47/I1/1

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