中文摘要：

目的探讨重组变异链球菌表面蛋白（rPAc）在大肠杆菌中可溶性表达的最佳诱导条件并制备其多克隆抗体。方法通过改变pET20b（＋）-AP/BL21（DE3）plysS工程菌的培养条件,提高rPAc可溶性表达水平。用纯化的rPAc免疫BALB/c小鼠制备多克隆抗体,通过酶联免疫吸附测定（ELISA）和Western blot法鉴定抗体的免疫学活性。结果pET20b（＋）-AP/BL21（DE3）plysS工程菌在Luria-Bertan（iLB）培养基（pH值为7.2）上培养,至表示菌体密度的光密度值OD600 nm=0.6时加入1.0 mmol.L-1异丙基-β-D-硫代吡喃半乳糖苷（IPTG）,30℃诱导培养4 h,rPAc的可溶性表达量最高。以纯化的rPAc免疫BALB/c小鼠,制备抗rPAc多克隆抗体,ELISA法测定抗体效价为1∶6 000,Western blot法鉴定出该抗体可特异性识别rPAc。结论 rPAc高效可溶性表达和抗rPAc多克隆抗体的制备为DNA初次免疫—蛋白质加强免疫策略的深入研究奠定了必要的物质基础。

英文摘要：

Objective The soluble protein recombinant Streptococcus mutans surface protein（rPAc） was expressed in Escherichia coli（E.coli） after the optimization of inducing conditions.The antiserum against rPAc was obtained by immunizing mice with the purified rPAc.Methods The soluble expression of rPAc in E.coli was further optimized by means of different culture conditions.Polyclonal antibody was made by immunizing mice with purified rPAc.Wes-tern blot and enzyme linked immunosorbent assay（ELISA） were carried out to identify the immunocompetence of the antibody.Results The highest soluble expression level of rPAc was obtained at Luria-Bertani（LB） medium（pH=7.2） when optical density（OD600 nm） was 0.6 after being induced at 30 ℃ for 4 h and the concentration of isopropyl β-D-1thigalactopyranoside（IPTG） was 1.0 mmol·L-1.The titer of the mice antiserum against rPAc was about 1∶6 000 by ELISA analysis,and rPAc could be specifically recognized by Western blot analysis.Conclusion This study proved that rPAc can be effectively expressed as a soluble form in E.coli,and the high specific polyclonal antibody of rPAc was proved to be prepared,which shed light on further research of DNA prime-protein boost inoculation.