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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2011, Vol. 31 Issue (06): 22-28    
研究报告     
重组PAC1-EC1(N)对表达不同PAC1变体的细胞系细胞活性的影响
李娟, 余榕捷, 王静静, 黄霖, 刘晓飞
暨南大学生物工程研究所 广州 510632
Biological Effects of Recombinant PAC1-EC1(N) on the Viability of Cell Lines with Different PAC1 Isoforms
LI Juan, YU Rong-jie, WANG Jing-jing, HUANG Lin, LIU Xiao-fei
Bioengineering Institute of Jinan University, Guangzhou 510632, China
 全文: PDF(874 KB)   HTML
摘要:

垂体腺苷酸环化酶激活多肽(pituitary adenylate cyclase-activating polypeptide,PACAP)特异受体PAC1(normal型)N端胞外域 具有调控PAC1活性的作用。为研究PAC1-EC1(N)对表达不同PAC1变体的细胞系活性的影响,利用基因工程技术获得C端带有6个his纯化标签的重组PAC1-EC1(N)。质谱检测、SDS-PAGE电泳和Western blot检测结果均显示成功表达和纯化目的蛋白PAC1-EC1(N)。Western blot检测确定兔眼角膜基质细胞、人神经母细胞瘤细胞SH-SY5Y、人前列腺癌细胞22RV1中分别表达一个或多个分子量不同的PAC1变体。重组PAC1-EC1(N)作用3株细胞,MTT方法检测细胞活性,结果显示:PAC1-EC1(N)有效促进只表达一个小分子PAC1变体的兔眼角膜基质细胞的增殖;但显著抑制只表达一个大分子PAC1变体的人神经母细胞瘤细胞SH-SY5Y的活性。对于表达3种大小不一的PAC1变体的人前列腺癌细胞22RV1,PAC1-EC1(N)对细胞的活性具有复杂的调控。显示PAC1-EC1(N)对表达PAC1变体数量不同,类型不同的细胞具有不同的生物活性的作用,提示了PAC1变体可能具有较复杂的自我调控机制。

关键词: 重组PAC1-EC1(N)胞外端细胞系生物活性    
Abstract:

PAC1 is a specific receptor for pituitary adenylate cyclase-activating polypeptide (PACAP).The N terminal first extracellular region of the PAC1 (PAC1-EC1) is involved in the regulation for the activation of PAC1. To study the effects of the EC1 domain of the PAC1 normal (N) isoform on the cell lines expressing different PAC1 isoforms, the recombinant protein PAC1-EC1 (N) with 6-His-tag at the C-terminus was expressed in an engineered Escherichia coli strain and purified by Ni-NTA affinity chromatography. Mass spectrum, SDS-PAGE and Western blot were used to characterize the recombinant PAC1-EC1(N). Western blot were used to identify the expression pattern of PAC1 isoforms in three cell lines including rabbit corneal stromal cells, human neuroblastoma cells SH-SY5Y and human prostate cancer cells 22RV1. MTT assays were used to detect different biological effects of the recombinant PAC1-EC1 (N) on the cell lines with different PAC1 isoforms. The results showed that PAC1-EC1(N) had significantly different biological effects on the cell lines with different PAC1 isoforms. PAC1-EC1(N) stimulated the viability of rabbit corneal stromal cells expressing only one PAC1 isoform with a smaller molecular weight, but inhibited the viability of human neuroblastoma cells SH-SY5Y expressing one type of PAC1 isoform with a larger molecular weight. Meanwhile, PAC1-EC1(N) caused complex biological effects on human prostate cancer 22RV1 cells with three types of PAC1 isoforms. These results showed that PAC1-EC1(N) exerts different effects on the viabilities of the cells with different expression patterns of PAC1 isoforms, indicating that the PAC1 owns a complex self-regulation mechanism of activation.

Key words: Recombinant PAC1-EC1 (N)    Extracellular region    Cell lines    Biological activity
收稿日期: 2011-03-03 出版日期: 2011-06-28
ZTFLH:  Q819  
基金资助:

国家自然科学基金 (30973244)、广东省科技攻关项目(2010B031200006)、重点高校研究基金(21609706, 21609407)资助项目

通讯作者: 余榕捷     E-mail: rongjie_yu1123@163.com
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引用本文:

李娟, 余榕捷, 王静静, 黄霖, 刘晓飞. 重组PAC1-EC1(N)对表达不同PAC1变体的细胞系细胞活性的影响[J]. 中国生物工程杂志, 2011, 31(06): 22-28.

LI Juan, YU Rong-jie, WANG Jing-jing, HUANG Lin, LIU Xiao-fei. Biological Effects of Recombinant PAC1-EC1(N) on the Viability of Cell Lines with Different PAC1 Isoforms. China Biotechnology, 2011, 31(06): 22-28.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I06/22

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