Prokaryotic Expression of i-type Lysozyme from Periplaneta americana and Preparation of Its Polyclonal Antibodies

doi:10.13560/j.cnki.biotech.bull.1985.2016.01.023

Abstract

Abstract: The objective of this work is to express i-type lysozyme(PaI)from Periplaneta americana in Escherichia coli and prepare its polyclonal antibodies of anti-PaI in mice. The coding gene of mature peptide region was amplified by PCR and prokaryotic expression vector pET28a-PaI was constructed. PaI-His fusion protein was expressed with IPTG induction and purified by Ni-NTA affinity chromatography. Then, Balb/c mice were immunized with the purified recombinant protein, and the titers of antiserum and the specificity of polyclonal antibodies were detected by indirect ELISA and Western blotting respectively. Results were as below. The length of nucleotide sequence encoding the mature peptide was 414 bp that encoded a putative protein with 137 amino acids. The constructed prokaryotic expression vector pET28a-PaI was successfully expressed in Escherichia coli. SDS-PAGE detection indicated that the PaI-His fusion protein was about 18 kD. Indirect ELISA and Western blotting analysis showed that the antiserum from immunized mice had high titer and specificity. In conclusion, the prokaryotic expression of PaI protein was successfully realized, and the anti-PaI polyclonal antibody with high efficiency and specificity were prepared, which laid the foundation for the further researches on the biological function of PaI protein.

Key words: Periplaneta americana, lysozyme, prokaryotic expression, polyclonal antibody

WANG Yun, ZHAI Su-zhen, ZHANG Chun-lin, WANG Ji-ping. Prokaryotic Expression of i-type Lysozyme from Periplaneta americana and Preparation of Its Polyclonal Antibodies[J]. Biotechnology Bulletin, 2016, 32(1): 138-143.

References

［1］Li L, Zhao J, Wang L, et al. Genomic organization, polymorphisms and molecular evolution of the goose-type lysozyme gene from Zhikong Scallop Chlamysfarreri［J］. Gene, 2013, 513(1)：40-52.
［2］Mitra P, Banerjee M, Biswas S, et al. Protein interactions of Merocyanine 540：spectroscopic and crystallographic studies with lysozyme as a model protein［J］. J Photochem Photobiol, B：Biology, 2013, 121：46-56.
［3］Huang P, Li WS, Xie J, et al. Charaterization and expression of HLysG2, a basic goose-type lysozyme from the human eye and testis［J］. Mol Immunol, 2011, 48(4)：524-531.
［4］Callewaert L, Michiels CW. Lysozyme in the animal kingdom［J］. J Biosci, 2010, 35(1)：127-160.
［5］Lapcharoen P, Komalamisra N, Rongsriyam Y, et al. Investigations on the role of a lysozyme from the malaria vector Anopheles dirus during malaria parasite development［J］. Dev Comp Immunol, 2012, 36(1)：104-111.
［6］Karthik V, Kamalakannan V, Thomas A, et al. Functional characterization of a c-type lysozyme from Indian shrimp Fenneropenaeus indicus［J］. Probiotics Antimicrob Proteins, 2014, 6(2)：114-121.
［7］Yue X, Liu B, Xue Q. An i-type lysozyme from the Asiatic hard clam Meretrix meretrix potentially functioning in host immunity［J］. Fish Shellfish Immunol, 2011, 30(2)：550-558.
［8］Buonocore F, Randelli E, Trisolino P, et al. Molecular characterization, gene structure and antibacterial activity of a g-type lysozyme from the European sea bass(Dicentrarchus labrax L.)［J］. Mol Immunol, 2014, 62(1)：10-18.
［9］Supungul P, Rimphanitchayakit V, Aoki T, et al. Molecular characterization and expression analysis of a c-type and two novel muramidase-deficient i-type lysozymes from Penaeus monodo［J］. Fish Shellfish Immunol, 2010, 28(3)：490-498.
［10］Wanniqama DL, Dwivede R, Zahraei-Ramazani A. Prevalence and antibiotic resistance of gram-negative pathogenic bacteria species isolated from Periplaneta americana and Blattella germanica in Varanasi, India［J］. J Arthropod Borne Dis, 2013, 8(1)：10-20.
［11］王赟, 龙高群, 张春林, 等. 美洲大蠊i型溶菌酶基因的克隆及其功能预测［J］. 动物医学进展, 2012, 33(9)：21-27.
［12］萨姆布鲁克 J, 拉塞尔 DW. 分子克隆实验指南［M］. 第3版. 黄培堂, 译. 北京：科学出版社, 2008.
［13］王荫长. 昆虫生物化学［M］. 北京：中国农业出版社, 2004：245-270.
［14］金小宝, 王艳, 朱家勇. 诱导前后美洲大蠊血淋巴抗菌活性的研究［J］. 广东药学院学报, 2006, 22(6)：665-682.
［15］李远辉, 李希, 冯建安, 等. 美洲大蠊不同提取部位的体外抑菌活性研究［J］. 中药与临床, 2014, 5(6)：27-29.
［16］Hata S, Ueno M, Kitamura F, et al. Efficient expression and purification of recombinant human m-calpain using an Escherichia coli expression system at low temperature［J］. J Biochem, 2012, 151(4)：417-422.
［17］才蕾, 矫丽媛, 王继华, 等. 甲胎蛋白的原核表达及复性优化［J］. 生物技术通报, 2015, 31(1)：193-197.
［18］高翠娥, 任晓宇, 张学尧, 等. 飞蝗CYP408B1和CYP409A1基因的原核表达［J］. 应用昆虫学报, 2015, 52(1)：153-161.
［19］孙卫国, 张灵霞, 杨秉芬, 等. 人血小板衍生生长因子BB的原核高表达及其包涵体复性［J］. 生物技术通讯, 2015, 26(1)：91-94.
［20］王倩, 杨眉, 冯瑞丽, 等. 人源DCF1基因原核表达载体构建及蛋白分离纯化［J］. 生物技术通报, 2014, 12：195-200.
［21］韩慧平, 于三科. 小鼠抗HCMV-gB多克隆抗体的制备及初步应用［J］. 动物医学进展, 2012, 33(2)：51-54.

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