含人FcγRⅡB基因慢病毒的制备及其在HT-1080细胞中的表达

Preparation of lentivirus containing human FcγRⅡB gene and FcγRⅡB expression in HT-1080 cells

目的构建FcγRⅡB慢病毒诱导表达载体,并鉴定其在细胞中的表达。方法以人mRNA为模板逆转录获得FcγRⅡB基因片段,并将其克隆入慢病毒表达质粒TRE,构建TRE-FcγRⅡB慢病毒表达重组质粒。将慢病毒表达重组质粒TRE-FcγRⅡB、慢病毒可诱导质粒Tet分别与慢病毒包装质粒共转染HEK293T细胞,分别包装表达病毒和可诱导病毒,分别测定表达病毒和可诱导病毒感染滴度。表达病毒和可诱导病毒共感染HT-1080细胞,经梯度浓度强力霉素(Dox)诱导,实时定量PCR检测FcγRⅡB mRNA表达,免疫荧光染色检测HT-1080细胞FcγRⅡB表达,Western blot法检测FcγRⅡB蛋白表达。结果重组质粒经PCR、酶切及测序鉴定,测序结果显示插入片段碱基序列与GenBank提供FcγRⅡB基因序列同源性100%。表达病毒滴度达106TU/mL,可诱导病毒滴度达105TU/mL,感染性良好。HT-1080细胞被病毒感染后经Dox诱导表达FcγRⅡB,免疫荧光染色结果显示FcγRⅡB能够在HT-1080细胞中表达;实时荧光定量PCR和Western blot法结果显示FcγRⅡB表达水平与诱导剂浓度正相关。结论成功制备了FcγRⅡB慢病毒,感染HT-1080细胞后可实现FcγRⅡB的诱导表达。

Objective To construct a lentiviral expression vector for FcγRⅡB and identify its expression in HT-1080 cells. Methods 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. Results The recombinant plasmid was identified using PCR assay and enzyme digestion analysis, and gene sequencing demonstrated that thenucleotide sequence of the inserted fragment had a homology of 100% with the Fc-yR II B nucleotide sequence provided by GenBank. The virus titer of the expression lentivirus was 106 TU/mL and the inducible lenUvirus was 105 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 Westem blotting showed that FcγRⅡB mRNA and protein expression levels were positively correlated with the concentration of Dox. Condusion The lentiviral expression vector for FcγRⅡB was successfully prepared and its expression in HT-1080 cells is controllable via the alterations of Dox concentration.