Liu Huiyu, Cao Xiuqin, Yang Zhiwei
Department of Pathogenic Biology and Immunology, School of Preclinical Medicine, Ningxia Medical University, Yinchuan 750004, China.
Key Laboratory of Fertility Preservation and Maintenase, Ministry of Education, Yinchuan 750004, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 Jun;30(6):561-4, 568.
To construct a lentiviral expression vector for FcγRIIB and identify its expression in HT-1080 cells.
FcγRIIB gene fragment was obtained using reverse transcription with human mRNA as the template, and then was cloned into TRE lentiviral expression plasmid to construct the lentiviral expression recombinant plasmid TRE-FcγRIIB. The recombinant plasmid TRE-FcγRIIB and lentiviral inducible plasmid Tet were transfected into HEK293T cells respectively with lentivirus packaging mix plasmids to pack the expression lentivirus and inducible lentivirus. The viral titers of the two lentiviruses were measured respectively. HT-1080 cells were coinfected with the expression lentivirus and the inducible lentivirus and induced by gradient-concentration doxycycline (Dox). The expression of FcγRIIB was detected by immunofluorescence technique. Real-time quantitative PCR (qRT-PCR) and Western blotting were used to detect FcγRIIB mRNA and protein expression levels, respectively.
The recombinant plasmid was identified using PCR assay and enzyme digestion analysis, and gene sequencing demonstrated that the nucleotide sequence of the inserted fragment had a homology of 100% with the FcγRIIB nucleotide sequence provided by GenBank. The virus titer of the expression lentivirus was 10(6) TU/mL and the inducible lentivirus was 10(5) TU/mL. The immunofluorescence technique showed that the HT-1080 cells co-infected by expression lentivirus and inducible lentivirus expressed FcγRIIB under the induction of Dox. The qRT-PCR and the Western blotting showed that FcγRIIB mRNA and protein expression levels were positively correlated with the concentration of Dox.
The lentiviral expression vector for FcγRIIB was successfully prepared and its expression in HT-1080 cells is controllable via the alterations of Dox concentration.
构建FcγRIIB慢病毒表达载体并鉴定其在HT - 1080细胞中的表达。
以人mRNA为模板，通过逆转录获得FcγRIIB基因片段，然后将其克隆到TRE慢病毒表达质粒中，构建慢病毒表达重组质粒TRE - FcγRIIB。将重组质粒TRE - FcγRIIB和慢病毒诱导质粒Tet分别与慢病毒包装混合质粒共转染入HEK293T细胞，包装表达慢病毒和诱导慢病毒。分别测定两种慢病毒的病毒滴度。HT - 1080细胞用表达慢病毒和诱导慢病毒共感染，并经梯度浓度强力霉素（Dox）诱导。采用免疫荧光技术检测FcγRIIB的表达。分别用实时定量PCR（qRT - PCR）和蛋白质印迹法检测FcγRIIB mRNA和蛋白表达水平。
通过PCR检测和酶切分析鉴定重组质粒，基因测序表明插入片段的核苷酸序列与GenBank提供的FcγRIIB核苷酸序列同源性为100%。表达慢病毒的病毒滴度为10(6) TU/mL，诱导慢病毒的病毒滴度为10(5) TU/mL。免疫荧光技术显示，表达慢病毒和诱导慢病毒共感染的HT - 1080细胞在Dox诱导下表达FcγRIIB。qRT - PCR和蛋白质印迹法显示，FcγRIIB mRNA和蛋白表达水平与Dox浓度呈正相关。
成功制备了FcγRIIB慢病毒表达载体，其在HT - 1080细胞中的表达可通过改变Dox浓度进行调控。
