【作者】 石建州；

【导师】 康相涛；

【作者基本信息】 河南农业大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 防御素是动物和植物体内抗感染及激发特异性抗感染免疫的重要抗菌肽。Gallinacin-4(Gal-4)是禽类体内重要的β-防御素。本试验主要是对不同禽类β-防御素基因的克隆、原核表达及其生物活性的鉴定。1、参照Genbank已发表的NM001001610(Gallinacin-4)序列,由其成熟肽片断(CDS除去signal peptide和propiece)设计引物,从红羽丝毛乌骨鸡、固始鸭、固始鹅、鸵鸟、鸽的骨髓中提取的RNA为模板,通过RT-PCR扩增出大小约为117bp的基因片段,并把它克隆到pUCm-T Vector上,构建重组质粒pUCm-Gal-4,经PCR、酶切鉴定及DNA测序验证,该基因Gal-4为编码序列,比较表明:五个品种Gal-4基因序列及推导氨基酸序列均为117bp,编码38个氨基酸,同源性达到100%;与AY621319报告的Gal-4基因序列完全符合,与NM001001610序列有一个碱基不同。分别登录注册Genbank。登录号为:红羽丝毛乌鸡DQ673442、鸽子DQ860106、鸵鸟EF031035、固始鸭EF606702、固始鹅EF606703。Gal-4基因的克隆成功为进一步研究禽类的β—防御素基因功能和应用奠定了基础。2、分析Gal-4成熟肽基因序列117bp(含一个终止密码子),含有5个稀有密码子用原核常用的密码子替换,重新合成117bp基因,其氨基酸仍为原来氨基酸序列。在以上基础上,我们把新合成的基因片段分别引入BamHI、HindⅢ酶切位点,亚克隆到同样双酶切的大肠杆菌原核表达载体pGEX-4T-1(BamHI和HindⅢ)中,构建重组表达质粒pGEX-4T-1-Gal-4,经PCR、酶切鉴定并进行确证性序列鉴定,结果表明,将Gal-4的基因正确地插入到原核表达载体pGLX-4T-1的目的位点,重组质粒pGEX-4T-1-Gal-4转化受体菌BL21(DE3)PlySs中,用IPTG诱导,经SDS-PAGE电泳显示,pGLX-4T-1-Gal-4表达产物分子质量约为30KDa,与理论推测的蛋白分子质量基本一致,进一步优化表达条件,1.0mmol/L IPTG在37℃诱导4小时条件下,表达量最大。3、重组蛋白的变性、复性研究。考虑到降解和表达产物对宿主菌的毒性作用的影响,用1.0mmol/L IPTG在37℃诱导4小时和25℃诱导2小时,其融合蛋白粗提物进行微量琼脂糖弥散法测定这种防御素的抗菌活性,对金黄色葡萄球菌(革兰氏阳性菌)、大肠杆菌(革兰氏阴性菌)和真菌(白色念珠菌)均无的抑制作用。其防御素的原核表达有待于进一步研究探讨。更多还原

【Abstract】 The experiment research Gallinacin-4 gene cloning,Escherichia coli expression and its bio-activity identification from poultry difference speciations.Defensins are imporce anti-bacterial peptides and anti-infect excitated specific exempt from conscription in animals and plants.Gallinacin-4(Gal-4)is importanceβ-defensin in vivo of birds.In order to acquire defensins peptides,experiment contruction gene procarryon ion carder and expression in Escherichia coli.The research refer to Genbank already deliver NM001001610 sequences, mature peptidc of NM001001610 sequences(CDS remove signal peptide and propiece)design primers,silly fowl、Gushi duck、Gushi goose、ostrich、dove extraction RNA form bone marrow,RT-PCR amplication about 117bp gene fragments,and clone pUCm-T Vector,establish pUCm-Gal-4 of recombinant plasmid, PCR、the enzyme cut identification and DNA gene sequence identification,This gene Gal-4 is code sequence,comparative indicate:five breeds Gal-4 gene sequences and deduction amino acid sequences all are 117bp,code 38 amino acid sequences, nucleotide homology arrive at 100%:AY621319 reportorial Gal-4 gene sequence complete accordance.NM001001610 sequence have only one base differece.(Gushi duck has one base mutation,but coded amino acid sequence no change.There were three differences of cause analyse:1.there might be speciations among themselves exist such mutation:2.PCR inflict bases mutation likelhood:3.there might be gene sequence exist error and so forth.)record registration Genbank,The accession number was:silky fowl DQ673442,dove DQ860106、ostrich EF031035、Gushi duck EF606702、Gushi goose EF606703.We get soluble expression products ofβ—defensins,which provides a sound basis for future research on its activity and purification..To lay the foundation of furher cloning,expressing the Gal-4 gene, further studying of the function of the Gal-4 gene prodct. Analysis of Gal-4 maturation peptides DNA sequence 114bp(contain one ending codons),five Rare Codons by Substitution of prokaryotic common. Codons.Afresh compound 114bp gene,amino acid sequence(38 entries)still amino acid sequence originally.Adhibit BamHI and SalIrestriction sites respectivel.Subclone similarly,using BamHI and SalI to double-cut p GEX-4T-lexpression vector and then link these two double-cut products together and transfer.Recombinant plasmid pGEX-Gal-4 transformed host strain BL21(DE3)PlySs,The expression was induced by IPTG.,SDS-PAGE electrophoretic display,pGEX-Gal-4 expressed protein was approximately 30KDa in molecular weight.,Results:The measured relative molecular mass was accorded with the theoretical value,The results of study on inducing and thecondition for Gal-4 protein expression was optimized. 1.0mmol/L,37℃,4 hours.The cloning and expression of the Gal-4 gene established the foundation of the further research about the function and application of the Gal-4 gene.This study lays a foundation for further research.The crude extricated substance of pGEX-4T-1-Gal fusion protein no inhibitory effect on Staphylococcus aureus、Escierichia Coli、funguses(Candida albicans)by agar diffusion test.更多还原