禽网状内皮组织增生症病毒CA蛋白的表达纯化及多克隆抗体制备

Expression and purification of reticuloendotheliosis virus CA protein and preparation of polyclonal antibody

根据禽网状内皮组织增生症病毒(REV)DBYR1102株的前病毒基因组c DNA序列设计并合成1对引物,以p T-G质粒为模板,通过PCR扩增获得该病毒CA基因片段。将其克隆于pET-30a(+)中,构建原核表达质粒pET30a-CA。重组质粒经双酶切和测序鉴定,转化BL21(DE3),经IPTG诱导,SDS-PAGE鉴定蛋白表达情况。利用Ni-NTA His Bind Resin纯化重组蛋白,并经蔗糖浓缩,Western blot法检测重组蛋白的反应原性。纯化后的目的蛋白免疫新西兰白兔,制备兔抗CA多克隆抗体并对其进行鉴定。结果表明构建的重组质粒pET-30a-CA在大肠杆菌BL21中呈可溶性高效表达。经Ni柱纯化和蔗糖浓缩后得到纯度较高的融合蛋白,以纯化的融合蛋白免疫新西兰白兔制备多克隆抗体,抗体效价达1∶25 600。该研究为REV的检测及CA基因的深入研究奠定了基础。

To express the CA protein of the reticuloendotheliosis virus（ REV）,the CA gene of REV DBYR1102 strain was cloned into prokaryotic expression vector pET-30a（ ＋）,and recombinant plasmid pET30a-CA was constructed. After double enzyme digestion and sequencing,the pET30a-CA was transformed into the Escherichia coli BL21（ DE3）. The transformants were induced by isopropylthio-β-Dgalactoside（ IPTG）,and SDS-PAGE was used to identify the expression of the CA protein. The His-tagged fusion protein REV-CA was ultrasonically treated and then purified through Ni-chelating affinity chromatography and sugar concentration. The purified CA was used to immune into New Zealand rabbit for preparing anti-CA polyclonal serum. The results showed that the recombinant plasmids pET30a-CA was successfully constructed,and the CA protein was expressed in soluble form. Polyclonal antibody was prepared and purified from immunized New Zealand white rabbit serum with the titer of 1 ∶ 25 600. This study laid the foundation for the development of the diagnostic methods and subunit vaccine for REV.