tPA/gGH双基因转染山羊乳腺上皮细胞表达分析

doi:10.16288/j.yczz.19-299

遗传 ›› 2020, Vol. 42 ›› Issue (4): 380-387.doi: 10.16288/j.yczz.19-299

tPA/gGH双基因转染山羊乳腺上皮细胞表达分析

宋绍征¹, 于康英¹, 张婷², 陆睿², 潘生强¹, 周鸣鸣¹(), 成勇²()

^1. 无锡太湖学院护理学院,基础医学系,无锡 214000
^2. 扬州大学兽医学院,江苏省转基因动物制药工程研究中心,扬州 225009

收稿日期:2019-09-24 修回日期:2019-11-29 出版日期:2020-04-20 发布日期:2020-02-27
通讯作者: 周鸣鸣,成勇 E-mail:zmm19770@126.com;chengyong12@yeah.net
作者简介:宋绍征,博士,讲师,研究方向：转基因与胚胎工程。E-mail: ssz0610@163.com
基金资助:
江苏省高校自然科学基金面上项目编号(17KJD310004);江苏省高校自然科学基金面上项目编号(19KJB180030);国家转基因生物新品种培育重大专项资助(2014ZX08008-004)

Expression analysis of tPA/gGH double genes in transfected goat mammary epithelial cells

Shaozheng Song¹, Kangying Yu¹, Ting Zhang², Rui Lu², Shengqiang Pan¹, Mingming Zhou¹(), Yong Cheng²()

^1. School of Nursing, Wuxi Taihu University, Wuxi 214000, China
^2. Jiangsu Provincial Research Center for Animal Transgenesis and Biopharming, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China

Received:2019-09-24 Revised:2019-11-29 Online:2020-04-20 Published:2020-02-27
Contact: Zhou Mingming,Cheng Yong E-mail:zmm19770@126.com;chengyong12@yeah.net
Supported by:
Supported by the Natural Science Foundation of Colleges and Universities in Jiangsu Province Nos(17KJD310004);Supported by the Natural Science Foundation of Colleges and Universities in Jiangsu Province Nos(19KJB180030);National Major Special Projects on New Cultivation for Transgenic Organisms No(2014ZX08008-004)

摘要/Abstract

摘要：

人组织纤溶酶原激活剂(tissue-type plasminogen activator, tPA)是一种被广泛应用于临床的溶栓药物。双基因共整合入生物体内能够产生协同作用,从而提高目的基因的表达水平。但是目前,利用gGH基因与tPA基因共整合以期提高tPA表达水平的相关研究较少。为筛选获得tPA高表达的tPA/gGH双基因整合的单克隆转基因山羊乳腺上皮细胞株,本研究以β-casein基因作为调控序列,构建乳腺特异性表达载体PCL25/gGH,并通过电转染将tPA和gGH双基因共转染山羊乳腺上皮细胞;通过G418筛选获得抗性细胞株,经PCR检测获得转基因单克隆细胞株;利用催乳素诱导tPA表达,收集48 h后细胞诱导液进行ELISA()和Western blotting检测并分析其tPA表达水平。结果表明,共获得142株抗性单克隆细胞,其中有53株tPA单基因整合细胞株,34株tPA/gGH双基因整合细胞株,双基因整合率达23.9% (34/142)。共检测出29株细胞能够表达tPA,其中单基因表达细胞为12株,表达率为22.6% (12/53);双基因表达细胞为17株,表达率为50.0% (17/34);且单基因细胞表达tPA含量为7.5~52.0 μg/mL,而双基因细胞表达tPA含量为40~360 μg/mL,明显高于单基因表达水平。本研究通过电转染的方式成功获得了tPA/gGH双基因整合的单克隆山羊乳腺上皮细胞株,并证明双基因整合的细胞株表达tPA水平明显提高,为后期制备高表达转基因山羊奠定了基础。

关键词: tPA, 双基因, 转基因, 山羊乳腺上皮细胞, 表达

Abstract:

tPA is a thrombolytic agent widely used in clinical settings. While double gene co-integration into organisms can produce synergistic effects and improved expression levels of the target gene, there are few reports detailing the co-integration of the tPA and gGH genes and an increased expression level of tPA. In order to study this, we obtained monoclonal goat mammary epithelial cell lines with tPA/gGH double gene integration and we analyzed the tPA expression level of single and double gene integration cells. We constructed a mammary gland-specific expression vector PCL25/gGH by using the β-casein gene as the regulatory sequence. The tPA and gGH genes were co-transfected into goat mammary epithelial cells by electrotransfection. Resistant cell lines were screened by G418, and transgenic monoclonal cell lines were obtained by PCR detection. tPA expression was induced by prolactin and subsequently, the cell induction solution was assayed after 48 hours by ELISA and Western blotting. The results show that a total of 142 resistant monoclonal cells were obtained including 53 tPA monogenic integration cell lines and 34 tPA/gGH double gene integration cell lines. The rate of double gene integration was 23.9% (34/142). A total of 29 cells were detected to be able to express tPA, of which 12 were single-gene-expressing cells and the corresponding expression rate was 22.6% (12/53). There were 17 double-gene- expressing cells with a corresponding expression rate of 50.0% (17/34). The expression level of tPA in single-gene cells was 7.5-52.0 μg/mL, while in double-gene cells was 40-360 μg/mL, which was significantly greater than that in single- gene cells. The goat mammary epithelial cell lines with tPA/gGH gene integration were successfully obtained by electrotransfection, and we proved that the expression level of tPA in the double gene integration cell lines with tPA/gGH gene integration was significantly increased. Our findings lay the foundation for the additional study of highly expressed transgenic goats and other animals with determination of scientific and clinical utility.

Key words: tPA, double genes, transgene, goat mammary epithelial cells, expression

宋绍征, 于康英, 张婷, 陆睿, 潘生强, 周鸣鸣, 成勇. tPA/gGH双基因转染山羊乳腺上皮细胞表达分析[J]. 遗传, 2020, 42(4): 380-387.

Shaozheng Song, Kangying Yu, Ting Zhang, Rui Lu, Shengqiang Pan, Mingming Zhou, Yong Cheng. Expression analysis of tPA/gGH double genes in transfected goat mammary epithelial cells[J]. Hereditas(Beijing), 2020, 42(4): 380-387.

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引物名称	引物序列(5°→3°)
gGH-F	CCGCTCGAGCGGATGATGGCTGCAGGCCCCCGGA
gGH-R	CCGCTCGAGCGGCTAGAAGGCACAGCTGGCCTCCCCG
CMV/tPA-F	CGTGGATAGCGGTTTGA
CMV/tPA-R	GAGCCCTCCTTTGATGC

tPA/gGH双基因转染山羊乳腺上皮细胞表达分析

Expression analysis of tPA/gGH double genes in transfected goat mammary epithelial cells

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参考文献 24

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细胞(142个)	整合细胞数	整合率(%)	表达细胞数	表达率(%)	表达水平(μg/mL)
tPA 单基因整合	53	37.3	12	22.6	7.5~52.0
tPA/gGH 双基因整合	34	23.9	17	50.0	40~360

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