大鼠Fcγ RⅡb基因慢病毒的制备及其在BMDC细胞中的表达检测

Preparation of Lentivirus of Rat FcγRⅡb Gene and Detection of Its Expression in BMDC Cells

目的构建大鼠FcγRⅡb慢病毒诱导表达载体,表达病毒与调节病毒共同感染大鼠骨髓来源树突状细胞(BMDCs)并检测FcγRⅡb的表达。方法提取大鼠肝脏mRNA,并以其为模板通过逆转录及PCR获得FcγRⅡb基因片段,利用酶切重连技术构建慢病毒表达载体TRE与FcγRⅡb重组慢病毒载体。使用HEK293T细胞分别包装TRE-FcγRⅡb表达病毒与TET-on调节病毒,并检测其病毒滴度;离体诱导培养大鼠骨髓来源未成熟树突状细胞并鉴定。采用OX-62免疫磁珠分选大鼠骨髓来源未成熟树突状细胞(i DC),用表达病毒和可诱导病毒共感染i DC。经一定梯度的强力霉素(DOX)诱导,分别用实时荧光定量PCR和蛋白免疫印记法(WB)检测BMDC中FcγRⅡb mRNA水平及FcγRⅡb蛋白表达水平;免疫荧光检测树突状细胞中FcγRⅡb的表达;电镜对分离培养的BMDC进行形态学观察和鉴定。结果成功构建TRE-FcγRⅡb重组慢病毒载体,经酶切、PCR电泳条带鉴定成功,基因序列测定结果与GenBank比对同源性100%。体外诱导培养BMDC,5d左右观察到明显的树突样凸起。成功用慢病毒感染BMDC后,FcγRⅡb mRNA和FcγRⅡb蛋白水平均较未感染BMDC有明显的增高。结论成功制备了含大鼠FcγRⅡb基因慢病毒,用其感染未成熟树突状细胞并经DOX诱导可实现FcγRⅡb的高表达。

Objective To construct a lentiviral expression vector for FcγRIIb,the expression plasmid and regulatory plasmid were co-transfectedto bone marrow-derived dendritic cells(BMDCs),then the expression of FcγRIIbwas detected.Methods Rat liver RNA was extracted and used as template to obtain FcγRIIb gene fragments by reverse transcription and PCR.Lentivirus expression vectors TRE and FcγRIIb were constructed by enzyme digestion and reconnection technology.HEK293T cells were used to package TRE-FcγRIIb expressing virus and TET-on regulating virus respectively,and their viral titers were detected.Immature dendritic cells derived from rat bone marrow were induced and cultured in vitro and identified.Immature dendritic cells(iDC)derived from rat bone marrow were isolated by OX-62 immunomagnetic beads.iDC was co-infected with the expressed virus and inducible virus.The expression of FcγRIIb mRNA level and FcγRIIb protein level in BMDC were detected by Real-time PCR and Western blotting after induction by doxycycline(DOX)with a certain gradient.The expression of FcγRIIb in dendritic cells was detected by immunofluorescence.The morphology of BMDCs was observed and identified by electron microscopy.Results TRE-FcγRIIb recombinant lentivirus vector was constructed.It was successfully identified through restriction enzyme digestion and PCR electrophoretic bands,with 100%homology of sequences.BMDCs were induced and cultured in vitro,and obvious dendritic protrusions were observed around the 5th day.After successful infection of BMDCs with lentivirus,the levels of FcγRIIb mRNA and FcγRIIB protein were significantly higher than those of uninfected BMDCs.Conclusion Up-regulation of rat BMDCs FcγRIIb expression can inhibit its antigen presentation function to some extent.