HLA-DR1二聚体的构建及其在昆虫细胞中的表达和纯化

Construction of HLA-DR1 Expression Vector and Its Expression in Insect Cells

目的构建HLA-DR1(由DRA和DRB1*01基因编码)杆状病毒表达载体,并使其在昆虫细胞内得到表达。方法采用RT-PCR方法从721.221细胞中扩增DRα和DRβ的信号肽及胞外序列,运用重叠PCR将DRα和DRβ片段分别与Fos和Jun的亮氨酸拉链序列相连,成为DRα-Fos和DRβ-Jun,DRα和DRβ可凭借Fos和Jun的亮氨酸拉链结合形成DR1分子,再通过EcoRⅠ酶切位点将DRα-Fos和人IgG1的Fc段相连,形成DRα-Fos-Fc重组序列,2个同源的DR1分子可通过IgG1的Fc段的二硫键结合形成二聚体。分别将DRα-Fos-Fc及DRβ-Jun插入杆状病毒表达载体pFastBacTMDual的2个多克隆位点处,构建出重组载体pFastBacTMDual+[DR1/Fc]。对构建的载体进行PCR及限制性内切酶酶切鉴定和测序。采用脂质体转染方法将表达载体转入昆虫细胞系Sf9中,通过双抗夹心ELISA及Westernblot检测HLA-DR1的表达。结果 PCR及酶切鉴定和测序证实目的基因插入正确且序列与GenBank一致。ELISA和Westernblot检测表明DR1杆状病毒感染的Sf9细胞培养上清HLA-DR1表达呈阳性,且表达的HLA-DR1分子具有正确的构象。结论成功构建出杆状病毒表达载体pFastBacTMDual+[DR1/Fc],并在Sf9细胞中得到表达,为研究HLA-DR1限制性的T细胞应答奠定了基础。

Objective To construct a baculovirus expression vector which carries the gene of HLA-DR1（coded by DRA,DRB101 genes）,and investigate the expression of HLA-DR1 dimer molecules in insect cells.Methods The signal and extramembranous domains of DRα and DRβ were amplified by RT-PCR method from total RNA of 721.221 cell which expresses HLA-DR1 molecule.DRα and DRβ segments were joined with the leucine zipper motifs of Fos or Jun segments by overlapping-PCR,respectively.DRα and DRβ chains were integrated into a DR1 molecule by the leucine zipper motifs of Fos and Jun segments.DRα-Fos was joined with the Fc segment of human IgG1 through the EcoR Ⅰ restriction site,therefore,two DR1 molecules were integrated into bivalent DR1 by disulfide bond of the Fc segment.DRα-Fos-Fc and DRβ-Jun segments were cloned into two multiple clone sites of baculovirus expression vector pFastBacTMDual,respectively.The orientation and sequence of the recombinant expression construct named pFastBacTMDual＋／[DR1/Fc／] was confirmed by PCR,enzyme digestion and DNA sequencing.The construct was transfected into the insect cell line Sf9 by lipofection and the expression of HLA-DR1 was detected by Sandwich ELISA and Western blot.Results The orientation and sequence of the recombinant expression construct were confirmed by PCR,enzyme digestion and matching GenBank.HLA-DR1 expression was found and HLA-DR1 molecule had a correct conformation in both supernatant and cell lysate of transfected Sf9 cells by ELISA and Western blot analysis.Conclusion A baculovirus expression vector pFastBacTMDual＋／[DR1/Fc／] has been constructed,confirming that the HLA-DR1 dimer molecule can be expressed in Sf9 cells.It will establish a foundation for studying HLA-DR1-restricted T cell response.