重组变异链球菌表面蛋白的可溶性表达及抗体制备
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[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-Bertani（LB）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。
[关键词] 变异链球菌表面蛋白； 原核表达； 抗体制备
[中图分类号] R 781 [文献标志码] A [doi] 10.3969/j.issn.1000-1182.2012.03.007
Optimizing expression and antibody preparation of recombinant Streptococcus mutans surface protein Jin Jie1，2, Fan Mingwen2, Li Yuhong2. （1. Dept. of Operative Dentistry and Endodontics, Stomatological Hospital of Hang-zhou, Hangzhou 310006, China; 2. Key Laboratory for Oral Biomedical Engineering of Ministry of Education, Wuhan University, Wuhan 430079, China）
[Abstract] 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-1-
thigalactopyranoside（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.
[Key words] Streptococcus mutans surface protein； prokaryotic expression； antibody preparation
变异链球菌是龋病的主要致病菌，其表面蛋白（Streptococcus mutans surface protein，PAc）参与介导细菌对牙面的黏附[1]。研究[2]表明：携带PAc主要
免疫活性区A-P片段的DNA防龋疫苗pCIA-P可有效诱导实验动物的特异性免疫应答。目前的研究[3-4]认为：以DNA疫苗pCIA-P进行初次免疫，再用蛋白质