鸡、猪BPI氨基端的基因克隆和鉴定

为了克隆杀菌/通透性增加蛋白(BP I)N端cDNA,构建重组体pM D 18-T-BP I,并进行序列鉴定,应用RT-PCR技术,参照G enB ank报道的预测序列,从鸡多形核白细胞(PM N)mRNA中扩增出杀菌/通透性增加蛋白(BP I)氨基端95个氨基酸的基因片段,并与pM D 18-T连接,构建BP I氨基端的基因重组体,进行酶切鉴定和序列测定;从猪PM N的总mRNA中扩增出BP I氨基端48个氨基酸的基因片段,进行测序鉴定。获得鸡BP I N端长度为285 bp的基因片段。序列分析证实该片段中有3个点突变,但均不在活性中心;获得猪BP IN端长度为146 bp的基因片段。成功克隆出BP I N端基因,经序列测定,确认为BP I基因,为进一步表达该基因奠定了基础。

Cloning and identification of chicken and pig BPI protein N-terminal fragment

cDNA of N-terminal fragment of chicken bactericidal/permeability-increasing protein(BPI) was cloned and the recombinant plasmid pMD18-T-BPI was constructed,then it was confirmed.According to the the prediction sequences of GenBank,the gene which encode N-terminal fragment of BPI protein was amplified by RT-PCR from mRNA that was extracted from the chicken polymorphon uclear neutrophils(PMN).The PCR product was cloned into the pMD18-T vector and the sequence was confirmed by restriction enzyme digestion and dideoxy-mediated-chain termination.The gene which encode N-terminal fragment of BPI protein was amplified by RT-PCR from mRNA that was extracted from the pig PMN.The 284 bp and 146 bp fragments were acquired.N-terminal sequences of BPI were successfully cloned.The results showed that the sequences are the gene of chicken and pig BPI.