【摘要】 为构建稳定表达绵羊肺腺瘤病毒(JSRV)受体透明质酸酶-2(Hyal-2)的NIH 3T3细胞系,本研究采用RT-PCR方法从绵羊瘤胃组织总RNA中扩增Hyal-2的cDNA,并引入标签多肽HA序列和限制性内切酶位点,将其克隆至真核表达质粒pcDNA3.1(+)中,构建重组质粒pcDNA-Hyal2-HA。将该重组质粒转染NIH 3T3细胞,并利用G418筛选、纯化,获得了稳定表达JSRV受体蛋白Hyal-2的NIH 3T3细胞系。qPCR、间接免疫荧光及western blot检测结果表明,该细胞系在传代至15代后,Hyal2-HA在NIH 3T3细胞中仍然能够稳定表达,并且主要分布于细胞膜上。JSRV囊膜蛋白诱导细胞转化试验显示,该蛋白的瞬时表达可以导致NIH 3T3细胞出现转化聚积灶,但失去了对稳定表达绵羊Hyal-2蛋白NIH 3T3细胞系的诱导转化作用,其作用机制尚待阐明。该细胞系的建立为进一步研究JSRV与其受体的相互作用致瘤机制奠定了基础。更多还原

【Abstract】 To study the relationship between Jaagsiekte retrovirus(JSRV) and receptor of JSRV, a NIH 3T3 cell line expressing sheep receptor protein(Hyal-2) of JSRV was established. The Hyal-2 cDNA was amplified with an HA tag by RT-PCR and cloned into the eukaryotic expressing vector pcDNA3.1(+). The resulting recombinant plasmid was transfected into NIH 3T3 cells. A cell line stably expressing Hyal2-HA, named as NIH 3T3-Hyal2-HA, was screened and colony-purified under the drug selection of G418. The expression of Hyal2-HA was confirmed by qPCR, indirect immunofluorescence assay and western blot. Results indicated that the recombinant Hyal2-HA was correctly and effectively expressed in the NIH 3T3-Hyal2-HA cell line and was mostly located in the membrane. The transient expression of JSRV envelope protein(Env) in 3T3 cells resulted in the multiple formation of transformed cell colonies but failed to induce such morphology change in NIH 3T3-Hyal2-HA cells, which implicated an inhibitory effect of sheep Hyal-2 on JSRV Env by an unknown machenism. This Hyal2-HA expressing cell line provided a useful in vitro platform for studying the relationship between JSRV and its receptor.更多还原