山羊白细胞介素-4成熟蛋白基因的原核表达及其生物学活性检测

畜牧兽医学报

山羊白细胞介素-4成熟蛋白基因的原核表达及其生物学活性检测

张晓燕^1,2，严若峰¹，徐立新¹，宋小凯¹，李祥瑞^1*

(1. 南京农业大学动物医学院，南京 210095； 2. 山西运城农业职业技术学院，运城 044000)

收稿日期:2012-03-22 出版日期:2012-10-25 发布日期:2012-10-25
通讯作者: 李祥瑞（1958-），教授，博士生导师，E-mail: lixiangrui@njau.edu.cn
作者简介:张晓燕（1983-），女，山西平陆人，讲师，硕士，主要从事分子免疫学及动物细胞因子的克隆与应用研究，E-mail: zxy09161021@126.com
基金资助:
国家“863”项目（2011AA10A211）；江苏高校优势学科建设工程资助项目

Expression of Goat Mature Interleukin-4 Gene in vitro and Bioactivity Detection of Recombinant Protein

ZHANG Xiao-yan^1,2, YAN Ruo-feng¹, XU Li-xin¹, SONG Xiao-kai¹, LI Xiang-rui^1*

（1. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;
2. Yuncheng Agricultural Vocationtechnical College of Shanxi, Yuncheng 044000, China）

Received:2012-03-22 Online:2012-10-25 Published:2012-10-25

摘要/Abstract

摘要： 本研究旨在进行山羊成熟白细胞介素-4的体外表达及重组蛋白生物学活性分析。运用PCR技术以pMD18-T-pGIL-4（pGIL-4为山羊IL-4全序列基因）为模板扩增成熟蛋白（mIL-4）基因，并构建pET-32a(+)-mGIL-4原核表达载体，IPTG诱导目的蛋白表达。使用改进的方法纯化目的蛋白后，采用淋巴细胞转化试验检测重组蛋白对山羊外周血T淋巴母细胞的增殖活性。经测序，mIL-4含339个核苷酸，编码112个氨基酸。表达的重组蛋白约为31.3 ku，主要以包涵体形式表达。通过改进纯化方法获得了高纯度的重组蛋白。试验证明，纯化、复性后的重组蛋白能显著刺激山羊外周血T淋巴母细胞的增殖，并表现出一定的剂量依赖性，8 μg·mL^-1增殖能力最强。本研究为山羊重组白细胞介素-4的规模化生产及作为佐剂的开发应用奠定了基础。

Abstract: The aim of the present study was to express goat mature interleukin-4 gene in E. coli and to test the bioactivity of the recombinant protein. The mature GIL-4 (mGIL-4) was amplified using pMD18-T-pGIL-4 (pGIL-4, goat IL-4 complete sequence) as template by PCR. The recombinant plasmid of pET-32a(+) containing mGIL-4 gene was transformed into E. coli BL21, and induced by IPTG. The recombinant protein expressed in E. coli was purified by modified method and refolded. Lymphocyte transformation test was performed to detect the bioactivity of recombinant protein to stimulate the proliferation of T lymphocytes in goat’s peripheral blood mononuclear cells (PBMC). Sequence analysis of mGIL-4 demonstrated an open reading frame (ORF) of 339 base pairs encoding for 112 amino acids. The recombinant protein was approximately 31.3 kD and was mainly existed in the inclusion body. Through improved purification methods, highly purified recombinant protein was obtained. The bioactivity test proved that purified recombinant protein was able to significantly stimulate the proliferation of goat peripheral blood T lymphoblasts in a dose-dependent manner and the recombinant protein of 8 μg·mL^-1 presented the highest stimulation. The data provided foundations for the scale production and the application as adjuvant of the goat recombinant interleukin-4.

中图分类号:

S852.4

张晓燕，严若峰，徐立新，宋小凯，李祥瑞. 山羊白细胞介素-4成熟蛋白基因的原核表达及其生物学活性检测[J]. 畜牧兽医学报, doi: .

ZHANG Xiao-yan, YAN Ruo-feng, XU Li-xin, SONG Xiao-kai, LI Xiang-rui. Expression of Goat Mature Interleukin-4 Gene in vitro and Bioactivity Detection of Recombinant Protein[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

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