Preparation and Titer Assay of Antiserum against Chitinase MpCHI786 from the Desert Insect Microdera punctipennis
Author of the article:LU Xueying, LI Jieqiong, ZHUANG Shuzhen, LIU Xiaoning, MA Ji*
Author's Workplace：(Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China)
Key Words：Microdera punctipennis; chitinase; DNA prime-protein boost; antiserum preparation; antibody titer
Abstract：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 prim-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 Mpchi86 was amplified by RT-PCR, and inserted into pET30avector to construct the recombinant expression plasmid pET30a-Mpchi786. Fusion protein His-MpCHI786 was expressed in E. coli BL21 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, eukaryotic 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, antiserum 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 pcDNA3-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 was produced by using DNA prime-protein boost immunization strategy.