【摘要】 使用总RNA提取试剂盒,从1月龄粤黄鸡骨髓细胞中分离提取总RNA,经过反转录PCR(RT＿PCR)扩增出鸡β＿防御素(Gal＿2)cDNA片段。PCR产物经凝胶回收纯化,加A尾后与载体pGEM＿TEasy相连,然后转化感受态细胞DH＿5α。在含X＿gal、IPTG的LA平板上筛选阳性克隆,经菌落PCR与提取质粒作限制性酶切鉴定后,对目的片段进行测序。结果表明RT＿PCR扩增出的cDNA片段碱基数为195bp,用Blast程序进行检索比较表明此扩增序列与Genbank中发表的鸡Gal＿2cDNA编码区序列100%相同。该cDNA编码64个氨基酸,包括信号肽、前片段和成熟肽,成熟肽由36个氨基酸残基组成。更多还原

【Abstract】 Total cellular RNA was purified from the bone marrow cells of 1-month-old Yuehuang chicken with the total RNA isolation kit. Then the chicken β-defensin(Gallicin-2) cDNA fragment was amplified by reverse transcription-polymerase chain reaction(RT-PCR). After gel purification and addition of the A-tail , the RT-PCR products were inserted into the pGEM-T Easy cloning vectors. Recombinant pGEM-T Gal-2 vector was transduced into competent cell DH-5α. Clones containing the DNA fragment of interest were screened on the LA agar plate with X-gal and IPTG. Restriction analysis and PCR were performed to identify the clone containing the DNA fragment of interest, then the cDNA fragment of interest was sequenced. The result showed that the obtained 195?bp DNA fragment is identical to the Gal-2 cDNA sequence registered in GenBank. The cDNA fragment code 64 amino acid residues., comprised of signal peptide, propiece and mature peptide with 36 amino acid residues.更多还原