中文摘要：

目的几丁质酶是昆虫重要的防御物质,本研究采用DNA-蛋白质联合免疫策略制备荒漠昆虫小胸鳖甲几丁质酶（MpCHI786）的抗血清,用于检测小胸鳖甲在外界不良条件下几丁质酶的变化。方法从小胸鳖甲成虫中提取总RNA,反转录后RT-PCR扩增Mpchi786 cDNA,构建原核表达载体pET30a-Mpchi786,在大肠杆菌BL21中用IPTG诱导表达获得融合蛋白His-MpCHI786。对融合蛋白切胶纯化后与弗氏佐剂混合作为抗原。另外,构建真核表达质粒pcDNA3.0-Mpchi786,对小鼠尾静脉高压注射,8 h后RT-PCR检测其在肝脏的瞬时表达。以pcDNA3.0-Mpchi786肌肉注射免疫小鼠3次后,再用His-MpCHI786融合蛋白加强免疫2次,DNA免疫和免疫结束后收集免疫小鼠的血清,ELISA和Western blot法检测抗血清效价和特异性。结果 RT-PCR结果显示尾静脉高压注射8 h后,pcDNA3.0-Mpchi786质粒在小鼠肝脏有特异性表达。Western blot检测表明,用His-MpCHI786融合蛋白作为抗原,pcDNA3.0-Mpchi786DNA质粒免疫3次的抗血清和His-MpCHI786融合蛋白加强免疫2次后的抗血清都显示了特异性反应条带。ELISA检测结果显示抗血清效价高达1∶204 800以上。结论利用DNA-蛋白质联合免疫法能够获得效价高、特异性的小胸鳖甲几丁质酶MpCHI786的抗血清。

英文摘要：

Objective Chitinase is one of the important defensive materials in insect. This study aims at preparation of the antiserum against chitinase MpCHI786 from the desert insect Microdera punctipennis by using DNA prime-protein boost immunization strategy to detect the changes of chitinase in M. punctipennis under adverse environmental conditions. Methods Total RNA was extracted from adult M. punctipennis, and then reverse transcribed to obtain the total cDNA. The cDNA of Mpchi786 was amplified by RT-PCR, and inserted into pET30a vector to construct the recombinant expression plasmid pET30a-Mpchi786. Fusion protein His-MpCHI786 was expressed in E. coli BI221 by IPTG induction. The fusion protein was recovered and purified from the SDS-PAGE gel and mixed with Freund＇s adjuvant and used as antigen for the following experiments. In addition, eukaryotie expression plasmid pcDNA3.0-Mpchi786 was constructed and injected into mice from the tail vein by high-pressure injection. The transient expression of the pcDNA3.0-Mpchi786 was detected in liver by RT-PCR after 8 h. For immunization assay, pcDNA3.0-Mpchi786 plasmid was used as DNA vaccine to inoculate mice for three times prior to the twice His-MpCHI786 protein boost immunization. After DNA immunization and the final immunization, antise- rum was collected and identified by Western blotting and ELISA to verify the specificity and titer, respectively. Results RT-PCR results showed that after 8 h of the tail vein high pressure injection, the eukaryotic expression plasmid peDNA3.0- Mpchi786 was specifically expressed in mouse liver. The results of Western blotting showed that, when His-MpCHI786 was used as antigen, the antiserum that immunized three times with pcDNA3.0-Mpchi786 plasmid and the final antiserum after two times protein boost immunization with His-MpCHI786 fusion protein showed a specific band. ELISA assay also showed that the titer for the final antiserum was over 1：204 800. Conclusion High titer and specific antiserum against MpCHI786 from M. punctipennis