文献类型：期刊文献

中文题名：两种报告基因在达摩凤蝶细胞克隆株RIRI-PaDe-2-C6中的表达

英文题名：Expression of Two Reporter Genes in Clonal Cell Line RIRI-Pa De-2-C6 Developed from Papilio demoleus Linnaeus(Lepidoptera: Papilionidae)

作者：刘志刚[1] 丁伟峰[1] 孙娜[1] 张欣[1] 李娴[1] 谢世聪[1] 冯颖[1]

第一作者：刘志刚

机构：[1]中国林业科学研究院资源昆虫研究所,国家林业局资源昆虫培育与利用重点实验室,云南昆明650224

年份：2018

卷号：31

期号：4

起止页码：31-37

中文期刊名：林业科学研究

外文期刊名：Forest Research

收录：CSTPCD;;Scopus;北大核心:【北大核心2017】；CSCD:【CSCD2017_2018】；

基金：中央级公益性科研院所基本科研业务费专项资助项目(CAFYBB2016ZD005);中国林业科学研究院资源昆虫研究所中央及公益性科研院所基本科研业务费专项资金(riricaf2012003M)资助

语种：中文

中文关键词：达摩风蝶;单细胞克隆;昆虫细胞系;口一半乳糖苷酶;分泌型碱性磷酸酶;重组杆状病毒

外文关键词：Papilio demoleus;single cell cloning;insect cell line;β-galactosidase;secreted alkaline phosphatase;re-combinant baculovirus

分类号：Q965.8

摘要：[目的]前期研究中,项目组从达摩凤蝶细胞系RIRI-Pa De-2中分离培养出单细胞克隆株RIRI-Pa De-2-C6,通过感染野生型苜蓿银纹夜蛾核型多角体病毒(Ac MNPV)发现克隆株RIRI-Pa De-2-C6对病毒敏感性高于原细胞系RIRI-Pa De-2。本研究将对达摩凤蝶单细胞克隆株RIRI-Pa De-2-C6的生物学特性和外源蛋白表达特性进行研究,并与原细胞系RIRI-Pa De-2进行比较,评价其用于外源蛋白表达的可行性。[方法]使用Bac-to-Bac杆状病毒表达系统构建重组β-半乳糖苷酶杆状病毒(Ac MNPV-Gal)和重组分泌型碱性磷酸酶杆状病毒(Ac MNPV-SEAP),分别侵染RIRI-Pa De-2和RIRI-Pa De-2-C6。在感染后的24、48、72、96、120、144、168 h检测2种重组蛋白的表达量,并对2个细胞系的形态学、生长曲线、倍增时间及核型进行分析和比较。[结果]表明:RIRI-Pa De-2和RIRI-Pa De-2-C6均可表达β-半乳糖苷酶(β-Gal)和分泌型碱性磷酸酶(SEAP),单细胞克隆株RIRI-Pa De-2-C6对重组β-Gal的表达水平显著高于原细胞系RIRI-Pa De-2(P<0.05),在接种Ac MNPV-Gal后96 h表达量达到最高。RIRI-Pa De-2-C6对重组SEAP的表达水平与RIRI-Pa De-2无显著差异(P>0.05)。显微观察发现,RIRI-Pa De-2-C6的细胞类型全部为梭形,比原细胞系RIRI-Pa De-2的细胞组成更加单一。RIRI-Pa De-2-C6的群体倍增时间为94.94 h,比原细胞系RIRI-Pa De-2的倍增时间(67.42 h)长。核型分析显示,RIRI-Pa De-2-C6的染色体数量呈正态分布,数目为21 82条,与RIRIPa De-2的染色体数目分布范围(48 97条)存在显著差异(P<0.05)。[结论]通过单细胞克隆方法获得的克隆株RIRI-Pa De-2-C6无论在外源蛋白表达以及基础生物学特性方面均有别于原细胞系RIRI-Pa De-2。
[Objective] In the previous research, a monoclonal cell line RIRI-PaDe-2-C6 was established from Pap- ilio demoleus cell line RIRI-PaDe-2 and it was found that the two cell lines could be infected by wild-type Autogra- pha californica muhiple nucleopolyhedrosis virus （AcMNPV）. Especially, RIRI-PaDe-2-C6 was susceptible to Ac- MNPV and exhibited a higher production of AcMNPV polyhedral per infected cell averagely compared to the parent RIRI-PaDe-2 cells. The aim of this study is to further understand the characteristics of RIRI-PaDe-2-C6 in express- ing exogenous genes. [ Method ] The Bac-to-Bac baculovirus expression system was used to establish recombinant baculovirus carrying β-galactosidase gene and secreted alkaline phosphatase （SEAP） gene. The RIRI-PaDe-2-C6 was infected with recombinant virus. The expression levels of the two recombinant proteins were detected at 24, 48, 72, 96, 120, 144, and 168 hours after infection and compared with RIRI-PaDe-2 cells. The methods of cells mor- phology analysis, growth analysis and chromosome analysis were used to obtain the biological characteristics of RIRI- PaDe-2-C6. [Result] RIRI-PaDe-2-C6 and RIRI-PaDe-2 could be infected by recombinant baculovirus. The ex- pression of β-galactosidase （β-Gal） was significantly higher than that of RIRI-PaDe-2 （P 〈 0.05 ）, but no signifi- cant difference in expression level of secreted alkaline phosphatase （SEAP） was observed between RIRI-PaDe-2-C6 and RIRI-PaDe-2 （P 〉 0.05）. All cells were spindle-shaped in RIRI-PaDe-2-C6 which was more homogeneous than RIRI-PaDe-2. The cell population doubling time of RIRI-PaDe-2-C6 was 94.94 hours which was longer than those of RIRI-PaDe-2 （67.42 hous）. The averages chromosome numbers of RIRI-PaDe-2-C6 was 52.26 ± 30.48 which was significant different from that of RIRI-PaDe-2 （73.19 ±24.27）. [ Conclusion] The differences in ex- pressing exogenous genes and biological characteristics are significant between clone cell line RIRI-PaDe-2-C6 and its parent cell line RIRI-PaDe-2.