人视黄醇结合蛋白4在杆状病毒系统中的表达及其多克隆抗体制备

Expression of human retinol-binding protein 4 in insect baculovirus system and preparation of its polyclonal antibody

旨在利用杆状病毒系统表达、制备人视黄醇结合蛋白(RBP4)并检测其免疫原性。将人RBP4基因片段及信号肽SS64片段亚克隆到杆状病毒转移载体pFastBac-dual(pFBd)中,获得相应的重组转移质粒;转化大肠杆菌菌株DH10bac,转座后经筛选获得重组穿梭质粒rbacmid,将重组穿梭质粒转染孔板培养的Sf9细胞,获得含人RBP4表达框的重组杆状病毒,经过扩增获得毒种。毒种感染对数生长期的Sf9细胞并表达人RBP4蛋白(I-RBP4),通过SDS-PAGE和Western blotting对表达蛋白进行检测和鉴定。用毒种感染悬浮培养的Sf9细胞制备一批RBP4蛋白,完成SDS、Western blotting的检测及少量的多抗制备。纯化重组蛋白并与E.coli重组人RBP4(E-RBP4)分别免疫家兔。实验结果,酶切鉴定及测序证实重组转移质粒构建正确;成功构建重组RBP4-bacmid;人RBP4蛋白在昆虫细胞获得高效表达。表达的RBP4蛋白可以分泌到培养基中,分子量约为23 kDa,经过计算表达量为100 mg/L;纯化蛋白免疫兔子制备了多抗血清,血清滴度为1∶100 000,高于原核表达的抗体滴度(1∶10 000),与人体提纯蛋白制备的抗体滴度相近。杆状病毒系统高效表达了人的RBP4蛋白,具有较好的抗原性,并获得高亲和力的抗血清,为下一步的人血RBP4检测试剂盒的制备打下了坚实的基础。

To prepare recombinant human retinol binding protein 4（RBP4） by using the baculovirus expression system and to detect its immunogenicity,the fusion DNA fragment of secretory signal peptide SS64 and human RBP4 gene was subcloned into a baculovirus transfer vector pFastBac-dual（pFBd）,and the corresponding recombinant transfer plasmid was transformed into E.coli strain DH10bac,after transposition recombinant shuttle bacmid was screened out.The logarithmic phase Sf9 cells were transfected with the recombinant bacmid and then the recombinant baculovirus containing hRBP4 expression box were generated.After amplification of recombinant baculovirus,the recombinant baculovirus seeds were obtained.To express human RBP4,logarithmic phase Sf9 cells were infected with the virus seeds and SDS-PAGE and Western blotting were used to detect and identify the expression.Finally,to prepare a batch of RBP4 protein,logarithmic phase Sf9 cells in suspension culture were infected with recombinant baculovirus seeds and the supernatant was harvested after 120 hours post-infection for purification.Finally for preparation of polyclonal antibody and evaluation of immunogenicity,the recombinant hRBP4 from insect cells and from E.coli were immunized rabbits.Restriction enzyme digestion and sequencing confirmed that the recombinant baculovirus transfer plasmid was constructed correctly,and subsequently recombinant RBP4-bacmid was generated successfully.SDS-PAGE and Western blotting analysis suggested that human RBP4 protein was highly expressed in Sf9 cells with the molecular weight of approximately 23 kDa.The recombinant RBP4 protein could be secreted into the medium efficiently,and the expression level was calculated amount of 100 mg/L.Finally the rabbit antiserum was harvested after recombinant RBP4 immunization,therein the titer of antiserum against baculovirus recombinant RBP4 is 1：100 000 whereas the titer of antiserum against E.coli recombinant RBP4 is only 1：10 000.Overall,human RBP4 was high efficiently expressed successfully with good antigenicity in baculovirus system,and high affinity antiserum was obtained.A solid foundation was laid for the next step of the preparation of human serum RBP4 detection kit.