摘要
【目的】为获得高启动效率的乳腺特异启动子,对荷斯坦奶牛、娟姗牛和奶水牛的β-酪蛋白基因启动子进行比较研究。【方法】对Genbank中所收录的荷斯坦奶牛、娟姗牛和水牛β-酪蛋白基因启动子序列进行比对分析,按照保守序列分别设计启动子区和3′端ploy A序列引物,同时设计人IFNα-2b基因和EGFP-Neo筛选序列引物并引入预先设计的酶切位点以方便载体构建。采集静脉血液,提取基因组DNA。PCR克隆β-酪蛋白基因启动子区和3′端ploy A区,同时以实验室保存质粒为模板克隆人IFNα-2b基因和EGFP-Neo筛选序列。测序正确后,将各片段按设计顺序依次插入p MD18-T骨架中,构建成p HSTBCNp-IFN,p JSBCNp-IFN和p SNBCNp-IFN乳腺特异表达载体。将各载体转染Bcap-37细胞,经G418筛选出稳定整合的转基因细胞系。用胰岛素(1 mg·L-1),转铁蛋白因子(1 mg·L-1),氢化可的松(1 mg·L-1)和PRL(250IU·L-1)联合对转基因细胞系进行诱导,然后用PCR、Western blotting、QRT-PCR技术和ELISA方法分别在m RNA水平和蛋白质水平检测IFNα-2b的表达。【结果】PCR后获得了荷斯坦奶牛、娟姗牛和水牛β-酪蛋白基因启动子区,长度分别为5 219、5 244和5 216 bp,其中包括了第一外显子,第一内含子和部分第二外显子,部分第二外显子的51个碱基编码17个氨基酸的信号肽序列。克隆了1 166 bp的ploy A区序列。最终将构建的3个乳腺特异表达载体转染Bcap-37细胞并经过G418筛选后,获得了3个转基因细胞系。经激素诱导后,PCR、Western blotting、QRT-PCR和ELISA检测发现3个转基因细胞系都表达了IFNα-2b基因,并且发现娟姗牛β-酪蛋白基因启动子在调控IFNα-2b基因的表达上效率较高,在m RNA水平和蛋白质水平上显著高于其他两个启动子(P<0.05)。【结论】娟姗牛β-酪蛋白基因启动子在调控外源基因表达上具有较高的效率,是具有应用前景的乳腺特异性启动子。所构建的表达载体为IFNα-2b转基因动物乳腺生物反应器的制备奠定基础。
【Objective】In order to obtain a high efficiency mammary gland specific promoter, in the present study, promote efficiency of β-casein gene promoters derived from Holstein cow, Jersey cow and water buffalo will be comparatively studied.【Method】β-casein gene promoter sequences of Holstein cow, Jersey cow and water buffalo in Genbank were comparatively analyzed, primers of promoter sequences and 3′ploy A sequences were designed according to the conservative fragment respectively, primers of the human IFN-2b gene and EGFP-Neo selective marker fragment were also designed. To facilitate the constructing expression vectors, appropriate restriction enzyme digestion sites were introduced into all the primers. Genome DNA was isolated from the leukocytes in venous blood samples. β-casein gene promoter fragments and 3′ploy A fragments were amplified by PCR using the genome DNA as templates. Human IFNα-2b gene and EGFP-Neo selective marker fragments were also amplified by PCR using the plasmids conserved in our lab. Sequencing analyses were carried out; making sure that all the fragments were correct. Then, all fragments were inserted into the p MD18-T backbone in the designed order. Finally, 3 mammary gland expression vectors, p HSTBCNp-IFN, p JSBCNp-IFN and p SNBCNp-IFN were constructed. Transgenic cell lines were generated by transfected 3 recombinant plasmids into Bacp-37 cells and G418 selected. Transgenic cell lines were induced by combined hormones including Insulin（1 mg·L-1）, Transferrin factor（1 mg·L-1）, Hydrocortisone（1 mg·L-1） and PRL（250 IU·L-1）. IFNα-2b expression levels were analyzed at mR NA and protein level by PCR, Western blot, QRT-PCR and ELISA.【Result】β-casein gene promoter and 3′ploy A fragments of Holstein cow, Jersey cow and water buffalo were obtained by PCR. The fragments lengths were 5 219, 5 244 and 5 216 bp respectively. In the promoter fragments, the first exon, first intron and the partial second exon were included. 51 nucleotides in the partial second exon encoding 17 amino acid signal peptide. For the 3′ploy A fragments were all 1 166 bp. After 3 transgenic expression vectors were generated, and transfected into Bcap-37 cells. After G418 screening, 3 positive transgenic cell lines were obtained successfully. PCR, Western blot, QRT-PCR and ELISA results showed that IFNα-2b was successfully expressed in these 3 transgenic cell lines when hormone induced. Transgenic cell lines contain p JSBCNp-IFN expression vector showed a significantly higher expression level than the other two at m RNA and protein levels（P〈0.05）.【Conclusion】Jersey cow β-casein gene promoter possesses a higher efficiency in regulating foreign gene expression, and it is a considerable mammary gland specific promoter in transgenic research. Expression vectors constructed in the present study laid a foundation for constructing IFNα-2b transgenic mammary gland bioreactor.
出处
《中国农业科学》
CAS
CSCD
北大核心
2016年第5期970-978,共9页
Scientia Agricultura Sinica
基金
国家"863"项目(2011AA100607)
国家转基因专项(2014ZX08007001-002)
