[1]高安键,赵若聪,罗伟芝,等.猪蛔虫抗菌肽Cecropin p基因的克隆及其原核表达载体的构建[J].江西师范大学学报(自然科学版),2012,(05):542-546.
 GAO An-jian,ZHAO Ruo-cong,LUO Wei-zhi,et al.The Cloning and the Construction of Expression Vector of Cecropin p from Ascaris Suum[J].,2012,(05):542-546.
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猪蛔虫抗菌肽Cecropin p基因的克隆及其原核表达载体的构建()
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《江西师范大学学报》(自然科学版)[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2012年05期
页码:
542-546
栏目:
出版日期:
2012-10-01

文章信息/Info

Title:
The Cloning and the Construction of Expression Vector of Cecropin p from Ascaris Suum
作者:
高安键;赵若聪;罗伟芝;魏鹏飞;刘志刚
深圳大学医学院过敏反应与免疫学研究所, 广东 深圳 518060
Author(s):
GAO An-jian ZHAO Ruo-cong LUO Wei-zhi WEI Peng-fei LIU Zhi-gang
关键词:
猪蛔虫Cecropin 2Cecropin 3Cecropin 4载体构建序列分析
Keywords:
ascaris suum Cecropin p2 Cecropin p3 Cecropin p4 cloning
分类号:
Q78
文献标志码:
A
摘要:
从猪蛔虫提取总RNA,反转录成cDNA后分别设计引物对Cecropin 2、Cecropin 3和Cecropin 4基因进行 PCR 扩增,分别得到带有双酶切位点的目的基因片段.将目的基因片段与 PMD18 T 载体连接,转化Top10克隆菌,测序正确后克隆入 pET28a 表达载体,进行双酶切和测序鉴定.研究结果表明,克隆得到的Cecropin p2、 Cecropin p3和Cecropin p4基因均由225个碱基组成,一个编码由74个氨基酸残基组成的开放阅读框,其蛋白相对分子质量约为8 kD,等电点分别为9.86、10.16和9.16.同源性分析结果显示:克隆所得猪蛔虫Cecropin p2、 Cecropin p3和Cecropin p4与数据库NCBI中的猪蛔虫Cecropin p2、Cecropin p3和Cecropin p4基因同源性均为100;,与其它Cecropin p基因同源性也较高(>42;).分子进化树显示, Cecropin p4的分化出现最早,其次为Cecropin p2, Cecropin p1和Cecropin p3分化得最晚.成功克隆了猪蛔虫Cecropin 2、Cecropin 3和Cecropin 4基因,构建了它们的原核表达载体并进行了生物信息学分析,为其重组表达和抑菌活性鉴定等研究奠定了理论基础.
Abstract:
Extracted the RNA from Ascaris suum and reverse-transcribed into cDNA. Ran PCR amplification and linked to PMD18 T vector, then transformed into E.coli Top10 and went on sequencing. cloned the gene into pET28a expression vector and made Restriction enzymes digestion analysis.The sequencing showed the cloned Cecropin 2, Cecropin 3, Cecropin 4 genes from Ascaris suum all contain 225 bp encoding an open reading frame of 74 amino acids with the deduced molecular weight around 8 kD and the isoelectric point 9.86、10.16、9.16 respectively. homology analysis indicated All 3 genes are of high homology with the sequences of corresponding Cecropin p genes registered on NCBI, and the differentiation of Cecropin p4 preceded that of the others showed by phylogenic tree. The genes of Cecropin2, Cecropin3 and Cecropin4 were cloned successfully, expression vectors were constructed and the sequence analysis including Homological analysis, phylogenic tree were done, all of these has paved the way for the protein expression and the test of antimicrobial activities.

参考文献/References:

[1] Boman H G. Antibacterieidal peptides: key components needed in immunity [J]. Cell, 1991, 65: 205-207.
[2] Jaynes J. Lyticpeptides, A magic bullet? [J]. J Biotechology News, 1990, 10(1): 8-10.
[3] Gabay J E. Ubiquitous natural antibiotics [J]. Science, 1994, 264(5157): 373-374.
[4] Steiner H, Hultmark D, Engstr?m A, et al. Sequence and specificity of two antibacterial proteins involved in insect immunity [J]. Nature, 1981, 292: 246-248.
[5] Hancock R E, Lehrer R. Cationic peptides: a new source of antibiotics [J]. Trends Biotechnol, 1998, 16(2): 82-88.
[6] Shin S Y, Kang J H, Jang S Y, et al. Effects of the hinge region of cecropin A(1-8)-magainin 2(1-12), a synthetic antimicrobial peptide, on liposomes, bacterial and tumor cells [J]. Biochim Biophys Acta, 2000, 1463(2): 209-218.
[7] Yedery R D, Reddy K V. Antimicrobiai peptides as microbicidal contraceptives: prophrcies for prophylactics [J]. Eur J Contracept Reprod Health Care, 2005, 10(1): 32-42.
[8] Andreu D, Rivas L. Animal antimicrobial peptides anoverview [J]. Biopolymers, l998, 47(6): 415-433.
[9] Zasloff M. Antibiotic peptides as mediators of innate immunity [J]. Curr Opin Immunol, 1992, 4(1): 3-7.
[10] 马卫明, 佘锐萍, 彭芳珍, 等. 猪肠道组织中抗菌肽类物质的分离提取及部分生物学活性研究 [J].科学技术与工程, 2004, 4(3): 202-205.
[11] 马卫明, 余锐萍, 胡艳欣, 等. 猪小肠抗菌肽对雏鸡的促生长作用及其机理初探 [J].中国农业科学, 2006, 39(8): 1723-1728.
[12] 罗伟芝, 符春荣, 邬玉兰, 等. 罗氏沼虾原肌球蛋白基因的克隆表达及变应原性鉴定 [J]. 江西师范大学学报: 自然科学版, 2012, 36(4): 425-430.
[13] DeLucca Delucca A J, Bland J M, Jacks T J, et al. Fungicidal activity of cecropin A [J]. Antimicrob Agents Chemother, 1997, 41(2): 481-483.
[14] Hultmark D, Steiner H, Rasmuson T, et al. Insect immunity: purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupa of Hyalophora cecropina [J]. Eur J Biochem, 1980, 106(1): 7-16.
[15] 崔勇, 仲维霞, 孔凡红, 等. 蛔虫抗菌肽Cecropin P1 基因在大肠埃希菌BL21(DE3)中的表达 [J]. 中国病原生物学杂志, 2009, 4(7): 532-551.

更新日期/Last Update: 1900-01-01