中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL VIROLOGY
2013年
6期
483-485
,共3页
曹守春%李玉华%李加%唐建蓉%石磊泰%刘景华%王云鹏%俞永新%董关木
曹守春%李玉華%李加%唐建蓉%石磊泰%劉景華%王雲鵬%俞永新%董關木
조수춘%리옥화%리가%당건용%석뢰태%류경화%왕운붕%유영신%동관목
狂犬病病毒%糖蛋白类%细胞系
狂犬病病毒%糖蛋白類%細胞繫
광견병병독%당단백류%세포계
Rabies virus%Glycoproteins%Cell lines
目的 构建能稳定表达狂犬病病毒糖蛋白的CHO细胞系.方法 用RT-PCR法扩增和分离CTN-1V株狂犬病毒糖蛋白基因,测序后克隆入pCDNA5.0FRT载体,构建重组质粒pCDNA5.0FRT-G,之后与POG44质粒共转染CHO细胞,采用潮霉素B抗性筛选,挑选阳性克隆,间接免疫荧光和Western Blot进行稳定表达细胞系的鉴定.结果 经酶切和测序鉴定,获得重组表达质粒pCDNA5.0FRT-G,共转染CHO细胞后,获得肉眼可见阳性细胞克隆,刮取阳性克隆进行传代扩大培养并定义为第2代,经过10代后免疫荧光和Western Blot结果依然阳性.结论 成功建立稳定表达狂犬病病毒糖蛋白的CHO细胞系,为深入研究糖蛋白的功能奠定基础.
目的 構建能穩定錶達狂犬病病毒糖蛋白的CHO細胞繫.方法 用RT-PCR法擴增和分離CTN-1V株狂犬病毒糖蛋白基因,測序後剋隆入pCDNA5.0FRT載體,構建重組質粒pCDNA5.0FRT-G,之後與POG44質粒共轉染CHO細胞,採用潮黴素B抗性篩選,挑選暘性剋隆,間接免疫熒光和Western Blot進行穩定錶達細胞繫的鑒定.結果 經酶切和測序鑒定,穫得重組錶達質粒pCDNA5.0FRT-G,共轉染CHO細胞後,穫得肉眼可見暘性細胞剋隆,颳取暘性剋隆進行傳代擴大培養併定義為第2代,經過10代後免疫熒光和Western Blot結果依然暘性.結論 成功建立穩定錶達狂犬病病毒糖蛋白的CHO細胞繫,為深入研究糖蛋白的功能奠定基礎.
목적 구건능은정표체광견병병독당단백적CHO세포계.방법 용RT-PCR법확증화분리CTN-1V주광견병독당단백기인,측서후극륭입pCDNA5.0FRT재체,구건중조질립pCDNA5.0FRT-G,지후여POG44질립공전염CHO세포,채용조매소B항성사선,도선양성극륭,간접면역형광화Western Blot진행은정표체세포계적감정.결과 경매절화측서감정,획득중조표체질립pCDNA5.0FRT-G,공전염CHO세포후,획득육안가견양성세포극륭,괄취양성극륭진행전대확대배양병정의위제2대,경과10대후면역형광화Western Blot결과의연양성.결론 성공건립은정표체광견병병독당단백적CHO세포계,위심입연구당단백적공능전정기출.
Objective To build the mammalian cell line of stable expression of rabies virus glycoprotein.Methods A amplified rabies virus glycoprotein gene of CTN-1V strain by RT-PCR method,and cloned into pCDNA5.0 FRT plasmid.Transfected the recombinant pCDNAS.0 FRT-G plasmid into CHO cells with POG44 together.Selection screens the positive cell with hygromycin B resistance.Identify the cell which stable express rabies virus glycoprotein with indirect immunofluorescence and Western Blot.Results Recombinant expression plasmid pCDNA5.0 FRT-G was successfully reconstruct by identification with enzyme digestion and sequencing.Get the visible positive cell clones,scraping into 96 well,it's the first passage.Subculture with 10 passages,the results of immunofluorescence and Western Blot were still positive.Conclusion Successfully established the stable expression of rabies virus glycoprotein CHO cell lines,and it's helpful to further study on the function of glycoprotein.
