Cloning，Sequence Analysis and Prokaryotic Expression of Musca domestica Allergen Tropomyosin Gene

Abstract

Abstract:

It was to probe into the homologous cloning of the Musca domestica allergen Tropomyosin gene, and then construct the prokaryotic expression vector which was expressed in Escherichia coli. Screening and isolation of Musca domestica Tropomyosin gene from Musca domestica cDNA library was carried out. The gene and encoded protein sequence of Tropomyosin was analyzed by bioinformatics methods , including general physical and chemical properties, signal peptide, secondary structure, tertiary structure, epitope and subcellular localization. Then plasmid pEASY-E1-Tropomysin were transformed into Escherichia coli BL21(DE3) competent cells for expression. The open reading frames of the Tropomyosin gene was 828 bp that encded a putative protein with 275 amino acids. The protein, with predicted molecular weight 31.6 kD and pI of 4.65, has the conserved Tropomyosin domain so belongs to Tropomyosin family. The result showed that the recombinant prokaryotic expression vector pEASY-E1-Tropomysin was successfully constructed and fusion protein was expressed in E. coli.

Key words: Musca domestic, Tropomyosin, Cloning, Bioinformatics, Allergen

Wei Chuanchuan, Xiu Jiangfan, Wang Yu, Guo Guo, Peng Chuanlin, Wu Qinyi, Wu Jianwei. Cloning，Sequence Analysis and Prokaryotic Expression of Musca domestica Allergen Tropomyosin Gene[J]. Biotechnology Bulletin, 2014, 0(8): 108-112.

References

［1］Rahuma N, Ghenghesh KS, Ben Aissa R, et al. Carriage by the housefly（Musca domestica）of multiple-antibiotic-resistant bacte-riat hat are potentially pat hogenic to humans, in hospital andother urban environment s in Misurata, Libya［J］. Ann Trop Med Parasitol, 2005, 99（8）：795-802.
［2］王继恒, 曲传智. 昆虫抗菌肽的研究进展［J］. 中国媒介生物学及控制杂志, 2000（5）：392-395.
［3］刘志刚, 邬玉兰, 杨睿, 吴海强. 家蚕变应原tropomyosin基因的克隆表达、纯化及其免疫学活性鉴定［J］. 免疫学杂志, 2009（6）：655-658.
［4］吉色曲伍. 疥螨原肌球蛋白基因的克隆、表达与蛋白定位研究［D］. 雅安：四川农业大学, 2011.
［5］黄素文, 杨文潮, 朱海, 等. 淡水小龙虾主要过敏原原肌球蛋白基因的克隆、表达及其免疫活性鉴定［J］. 中国免疫学杂志, 2011（7）：642-647.
［6］刘志刚, 何毅华, 吴海强, 朱永峰. 美洲大蠊Per a7基因的克隆、表达及免疫学鉴定［J］. 昆虫学报, 2007（4）：330-334.
［7］刘志刚, 何毅华, 高波. 德国小蠊变应原Bla g2的克隆、表达及其免疫学特性鉴定［J］. 中国人兽共患病学报, 2007（3）：248-251.
［8］陈红娜, 褚宏亮. 蟑螂变应原的研究进展［J］. 江苏预防医学, 2013（2）：44-45.
［9］李荔, 陈樱麟, 闫浩, 刘志刚. 斑马鱼原肌球蛋白基因克隆表达及免疫学鉴定［J］. 水产科学, 2013（1）：26-30.
［10］马慧, 刘志刚. 蟑螂重组变应原的研究进展［J］. 热带医学杂志, 2007（3）：288-291.
［11］Satinover SM, Reefer AJ, Pomes A, et al. Specific IgE and IgG anti-body-binding patterns to recombinant cockroach allergens［J］. The Journal of Allergy and Clinical Immunology 2005, 115（4）：803-809.
［12］Zhou B, Ensell M, Zhou Y, et al. Prevention and treatment of DNA vaccine encoding cockroach allergen Bla g 1 in a mouse model of allergic airway inflammation［J］. Allergy, 2012；67：166-174.
［13］Cheng XY, Altosaar L. High throughput screening method for identifying molecules having biocail function：USA, 20060068397［P］. 2004-09-24.
［14］刘红美, 张洁, 王贇, 等. 热胁迫后家蝇幼虫cDNA文库构建与随机EST测序分析［J］. 免疫学杂志, 2010（9）：772-775.
［15］金小宝, 王婷婷, 朱家勇. 家蝇幼虫脂肪体cDNA文库的构建及初步鉴定［J］. 中国人兽共患病杂志, 2005（1）：52-55.
［16］张帅, 崔金杰, 王春义, 等. 棉铃虫幼虫唾液腺cDNA文库的构建及EST分析［J］. 昆虫学报, 2012（6）：625-633.
［17］张道伟, 曾燕玲, 魏福伦, 周德贵. 二斑叶螨cDNA文库的构建及鉴定［J］. 广东农业科学, 2013（10）：149-151.
［18］李滨, 欧阳碧函, 徐宏博, 等. 刺毛虫毒刺cDNA文库的构建及初步分析［J］. 中国病原生物学杂志, 2011（11）：828-830, 836, 886.
［19］柯丹, 刁庆春, 邓抒琴. 重庆地区2210例过敏性皮肤病患者过敏原检测分析［J］. 现代医药卫生, 2007（4）：479-480.
［20］孙坚, 周世良, 漆兰, 等. 儿童过敏原皮肤点刺试验53例分析［J］. 实用临床医学, 2008（12）：90-92.
［21］Jeong KY, Hwang H, Lee J, et al. Allergenic characterization of tro-
pomyosin from the dusky brown cockroach, Periplaneta fuligi-nosa［J］. Clin Diagn Lab Immunol, 2004, 11（4）：680-685
［22］Leung PSC, Chu KH. cDNA cloning and molecular identification of the major oyster allergen from the Pacific oyster Crassostrea gigas.［J］. Clin Exp Allergy, 2001, 31（8）：1287-1294.
［23］Jeong KY, Hong CS, Yong TS. Allergenic tropomyosins and their cross-reactivities［J］. Protein Pept Lett, 2006, 13（8）：835-845.
［24］杜振彩. 昆虫致敏原改法制备后生物效应的比较［J］. 黑龙江中医药, 2002（6）：49-50.

[1]	LYU Qiu-yu, SUN Pei-yuan, RAN Bin, WANG Jia-rui, CHEN Qing-fu, LI Hong-you. Cloning, Subcellular Localization and Expression Analysis of the Transcription Factor Gene FtbHLH3 in Fagopyrum tataricum [J]. Biotechnology Bulletin, 2023, 39(8): 194-203.
[2]	WANG Jia-rui, SUN Pei-yuan, KE Jin, RAN Bin, LI Hong-you. Cloning and Expression Analyses of C-glycosyltransferase Gene FtUGT143 in Fagopyrum tataricum [J]. Biotechnology Bulletin, 2023, 39(8): 204-212.
[3]	SUN Ming-hui, WU Qiong, LIU Dan-dan, JIAO Xiao-yu, WANG Wen-jie. Cloning and Expression Analysis of CsTMFs Gene in Tea Plant [J]. Biotechnology Bulletin, 2023, 39(7): 151-159.
[4]	ZHANG Lu-yang, HAN Wen-long, XU Xiao-wen, YAO Jian, LI Fang-fang, TIAN Xiao-yuan, ZHANG Zhi-qiang. Identification and Expression Analysis of the Tobacco TCP Gene Family [J]. Biotechnology Bulletin, 2023, 39(6): 248-258.
[5]	LI Jing-rui, WANG Yu-bo, XIE Zi-wei, LI Chang, WU Xiao-lei, GONG Bin-bin, GAO Hong-bo. Identification and Expression Analysis of PIN Gene Family in Melon Under High Temperature Stress [J]. Biotechnology Bulletin, 2023, 39(5): 192-204.
[6]	GUO San-bao, SONG Mei-ling, LI Ling-xin, YAO Zi-zhao, GUI Ming-ming, HUANG Sheng-he. Cloning and Analysis of Chalcone Synthase Gene and Its Promoter from Euphorbia maculata [J]. Biotechnology Bulletin, 2023, 39(4): 148-156.
[7]	WANG Yi-qing, WANG Tao, WEI Chao-ling, DAI Hao-min, CAO Shi-xian, SUN Wei-jiang, ZENG Wen. Identification and Interaction Analysis of SMAS Gene Family in Tea Plant（Camellia sinensis） [J]. Biotechnology Bulletin, 2023, 39(4): 246-258.
[8]	YANG Lan, ZHANG Chen-xi, FAN Xue-wei, WANG Yang-guang, WANG Chun-xiu, LI Wen-ting. Gene Cloning, Expression Pattern, and Promoter Activity Analysis of Chicken BMP15 [J]. Biotechnology Bulletin, 2023, 39(4): 304-312.
[9]	YANG Jun-zhao, ZHANG Xin-rui, ZHAO Guo-zhu, ZHENG Fei. Structure and Function Analysis of Novel GH5 Multi-domain Cellulase [J]. Biotechnology Bulletin, 2023, 39(4): 71-80.
[10]	CHEN Qiang, ZHOU Ming-kang, SONG Jia-min, ZHANG Chong, WU Long-kun. Identification and Analysis of LBD Gene Family and Expression Analysis of Fruit Development in Cucumis melo [J]. Biotechnology Bulletin, 2023, 39(3): 176-183.
[11]	LIU Si-jia, WANG Hao-nan, FU Yu-chen, YAN Wen-xin, HU Zeng-hui, LENG Ping-sheng. Cloning and Functional Analysis of LiCMK Gene in Lilium ‘Siberia’ [J]. Biotechnology Bulletin, 2023, 39(3): 196-205.
[12]	PING Huai-lei, GUO Xue, YU Xiao, SONG Jing, DU Chun, WANG Juan, ZHANG Huai-bi. Cloning and Expression of PdANS in Paeonia delavayi and Correlation with Anthocyanin Content [J]. Biotechnology Bulletin, 2023, 39(3): 206-217.
[13]	XING Yuan, SONG Jian, LI Jun-yi, ZHENG Ting-ting, LIU Si-chen, QIAO Zhi-jun. Identification of AP Gene Family and Its Response Analysis to Abiotic Stress in Setaria italica [J]. Biotechnology Bulletin, 2023, 39(11): 238-251.
[14]	YANG Xu-yan, ZHAO Shuang, MA Tian-yi, BAI Yu, WANG Yu-shu. Cloning of Three Cabbage WRKY Genes and Their Expressions in Response to Abiotic Stress [J]. Biotechnology Bulletin, 2023, 39(11): 261-269.
[15]	CHEN Chu-yi, YANG Xiao-mei, CHEN Sheng-yan, CHEN Bin, YUE Li-ran. Expression Analysis of the ZF-HD Gene Family in Chrysanthemum nankingense Under Drought and ABA Treatment [J]. Biotechnology Bulletin, 2023, 39(11): 270-282.