摘要
根据GenBank公布的人和牛BMP4基因序列设计引物,通过RT-PCR和PCR方法分别扩增全长编码序列和内含子2、3,进行克隆及测序鉴定;根据已得到的序列设计引物扩增BMP4成熟蛋白cDNA,克隆入pET32a表达载体,转化E.coliBL21(DE3)plysS进行诱导表达,同时进行诱导条件的优化。结果显示,山羊BMP4基因全编码序列、内含子2和3序列长度分别为1 230、1 053、962 bp,与猪、人、小鼠和牛相应序列的同源性分别为:96.10%、94.96%、91.79%、99.19%,83.80%、76.03%、66.94%、96.71%和87.15%、80.24%、67.20%、98.13%。在E.coliBL21(DE3)plsS中表达的BMP4融合蛋白的相对分子量为31 000,适宜的诱导条件为:0.5 mmol/L IPTG、22℃、诱导8 h.结果表明,黄淮山羊BMP4基因全长编码序列和内含子2、3已被成功克隆。适宜表达条件的确定为进一步研究山羊BMP4的生物学功能奠定了基础。
Specific primers were designed according to the sequences of human and bovine BMP4 genes published on GenBank to clone the encoding sequence,intron2,and intron 3 of BMP4 gene in Huanghuai goat.In addition,the cDNA encoding the mature protein of goat BMP4 was amplified from the full coding DNA sequence,then it was cloned into the expression vector pET32a,and transformed into the host cell E.coli BL21（DE3）plysS.The inductive condition for expression was optimized.The results showed that the length of encoding sequence,intron2 and 3 of goat BMP4 were 1 230 bp,1 053 bp and 962 bp,respectively.Corresponding sequence comparisons of goat with those of pig,human,mouse and cattle demonstrated that the homologies of the nucleotides were 96.10%,94.96%,91.79% and 99.19% for the coding DNA sequences;83.80%,76.03%,66.94% and 96.71% for the intron 2 and 87.15%,80.24%,67.20%,98.13% for the intron 3,respectively.The molecular weight of goat BMP4 fusion protein was 31 KD,and the optimized condition for the inductive expression was at 22℃,0.5 mmol/L IPTG,for 8h induction.Thses results suggested that the full-length encoding sequence and the sequences of intron2 and intron3 of goat BMP4 gene have been successively cloned.The determination of optimized condition for prokaryotic expression supplies a basis for the further studies on the biological role of goat BMP4.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2010年第10期1363-1367,1376,共6页
Chinese Journal of Veterinary Science
基金
河南省教育厅自然基金资助项目(2007230003)
河南省科技攻关项目(0524030011)
