Cloning and prokaryotic expression of Anopheles sinensis defensin gene and preliminary bioactivity evaluation of the recombinant product

Abstract

Abstract: Objective To clone the defensin gene of Anopheles sinensis for its prokaryotic expression and preliminary bioactivity evaluation of the recombinant protein. Methods Two pairs of primer were designed and synthesized based on the published defensin sequences, and PCR amplification was performed using the cDNA of An.sinensis as the template. The PCR products were cloned and sequenced. A truncated defensin fragment was cloned into pET-32(a) plasmid and expressed with the induction by IPTG. The expressed product was purified and its bioactivity evaluated with agarose diffusion assay. Results The PCR product 270 bp in size was identified as the coding sequence for defensin after sequence analysis, which had 85% homology with the defensin sequence from An.gambiae. The truncated defensin (162 bp) cloned was subcloned into the expression plasmid pET-32(a) and the molecular weight of the target fusion protein was 26 000 after IPTG induction. Preliminary experiment, however, failed to demonstrate bacteriostatic effect of the purified recombinant protein. Conclusion The coding sequence for defensin gene of An.sinensis has been successfully cloned and the truncated fraction can be highly expressed as a soluble fusion protein, which, however, does not possess bacteriostatic activity.

CLC Number:

YAN Xiao-yu, CHEN Xiao-guang, PENG Hong-juan, ZHENG Xue-li. Cloning and prokaryotic expression of Anopheles sinensis defensin gene and preliminary bioactivity evaluation of the recombinant product[J]. Journal of Southern Medical University, 2005, 25(04): 371-376.

References

[1] Matsuyama K, Natori S. Purification of three anti-bacterial proteins from the culure medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina[J]. J Biol Chem, 1988, 263(32): 17112-6.
[2] Chalk R, Townson H, Natori S, et al. Purification of an insect defensin from the mosquito, Aedes aegypti[J]. Insect Biochem Mol Biol, 1994, 24(4): 403-10.
[3] Eggleston P, Lu W, Zhao Y. Genomic organization and immune regulation of the defensin gene from the mosquito, Anopheles gambiae[J]. Insect Mol Biol, 2000, 9(5): 481-90.
[4] 支国舟,陈晓光,周晓红,等.蚊防御素基因的克隆及序列分析[J] 第一军医大学学报,2002,22(6):499-502.Zhi GZ, Chen XG, Zhou XH, et al. Cloning and sequence analysis of mosquito defensis genes[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2002, 22(6): 499-502.
[5] 刘先凯,赵彤岩,朱礼华,等.白纹伊蚊和埃及伊蚊defensinA基因克隆及序列分析[J].寄生虫与医学昆虫学报,2002,9(4):216-21.Liu XK, Zhao TY, Zhu LH, et al. Cloning and sequencing of the defensis A gene from Aedes aegypti and Ae. albopictus[J]. Acta Parasitol Med Entomol Sin, 2002, 9(4): 216-21.
[6] 刘先凯,赵彤岩,朱礼华,等.白纹伊蚊和埃及伊蚊防御素(defensin)成熟肽基因克隆及结构推测[J].寄生虫与医学昆虫学报,2003,10(1):30-4.Liu XK, Zhao TY, Zhu LH, et al. Gene cloning of mosquito defensin mature peptide and conjecture of the secondary and tertiary structure[J]. Acta Parasitol Med Entomol Sin, 2003, 10(1): 30-4.
[7] Cho WL, Fu TF, Chiou JY, et al. Molecular characterization of a defensin gene from the mosquito[J]. Insect Biochem, 1997, 27(5):351-8.
[8] 姜泊,张亚历,周殿元.分子生物学常用实验方法[M].北京:人民军医出版社,1996.53-4.
[9] 陈奇.中药药理研究方法学[M].北京:人民卫生出版社,1993259-65.
[10] Bals R. Epithelial antimicrobial peptides in host defense against infection[J]. Respir Res, 2000, 1(3): 141-50.
[11] Ortega MR, Ganz T, Milner SM. Human beta defensin is absent in burn blister fluid[J]. Burns, 2000, 26(8): 724-7.
[12] Saberwal G, Nagaraj R. Cell-lytic and antibacterial peptides that act by perturbing the barrier function of membranes: facets of their conformational features, structure-function correlations and membrane-perturbing abilities[J]. Biochim Biophys Acta, 1994,1197(2): 109-31.

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