大熊猫秦岭亚种白细胞介素-2的克隆、体外表达及生物学活性研究

[Study on the cloning, in vitro expression and bioactivity of Ailuropoda melanoleuca Qinling subspecies interleukin-2].

作者信息

Qiao Qi, Ma Qing-yi, Zhu Hui, Chen De-kun

机构信息

College of Veterinary Medcine, Northwest A & F University, Yangling 712100, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2009 Jun;25(6):479-82.

PMID:19500497

Abstract

AIM

To identify if any difference exisit between the population of Sichuan and Qinling giant panda. express IL-2 protein in BL21(DE3)with biological activity.

METHODS

IL-2 was amplified by RT-PCR from adult Qinling giant panda peripheral blood lymphocyte which was induced by ConA, and cloned into prokaryotic expression vector of pET32a-IL-2. The fusion protein was expressed in BL21(DE3)/pET system and identified by SDS-PAGE and Western blot; inoculate rabbit with purified expression products to prepare polyclonal antibody; use lymphocytes proliferation in vitro to detect biological activity.

RESULTS

The protein of IL-2 was obtained by recombination expression, molecule weight is 34 000 .The specificity of polyclonal antibody was obtained, in vitro activity test indicated that the recombinant protein IL-2 having an activity of promoting the proliferation of lymphocytes. The effect can be stopped by polyclonal antibody which was prepared before.

CONCLUSION

The IL-2 gene of Qinling giant panda was cloned and expressed in E.coli successfully, and the homology of IL-2 gene in these two population is 99.4% and the recombination protein can promote the proliferation of lymphocytes in vitro from giant panda.

摘要

目的

鉴定四川大熊猫种群和秦岭大熊猫种群之间是否存在差异，在BL21(DE3)中表达具有生物学活性的白细胞介素-2（IL-2）蛋白。

方法

从经刀豆蛋白A（ConA）诱导的成年秦岭大熊猫外周血淋巴细胞中通过逆转录聚合酶链反应（RT-PCR）扩增IL-2，并克隆到原核表达载体pET32a-IL-2中。融合蛋白在BL21(DE3)/pET系统中表达，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blot）进行鉴定；用纯化的表达产物免疫家兔制备多克隆抗体；采用体外淋巴细胞增殖实验检测生物学活性。