摘要
将含有蚓激酶基因的表达盒插入到逆转录病毒载体pLNCL中获得了蚓激酶重组逆转录病毒载体,转染泌乳期奶牛乳腺小叶并获得暂时表达。利用脂质体转染PA317包装细胞,G418进行筛选得到了阳性细胞克隆,克隆扩大培养后病毒上清感染NIH3T3细胞进行滴度测定,最高滴度为1×104CFU/mL。进而用病毒上清转染妊娠期奶牛乳腺小叶组织,产后奶样经FAPA法检测表明蚓激酶基因已经整合到奶牛乳腺组织,并能实现相对稳定的表达。
Lumbrokinase （LK） gene was cloned into the retroviral vector pLNCL to construct the recombinant retroviral vector pLN-bCP-LK. The result of FAPA showed that LK gene had been expressed in the milk by injecting the plasmid into the galactophore of cow. The vector were transfected into PA317 cells for packaging by liposomal transfection,after G418 selection for 2 weeks,G418-resistant PA317 colonies were obtained and amplified. The supernatant of cell culture was harvested and infected NIH3T3 cells,as were to measure the viral titer of recombinan retrovirus. The results showed that the highest titer of viral supernatant was 1 × 10^4 CFU/mL. The supernatant of cell culture was injected into the mammary gland of pregnant cow. The result of FAPA showed that LK gene had been expressed continuously in the milk of the post partum cow.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2006年第5期519-521,共3页
Chinese Journal of Veterinary Science
基金
国家"863"高技术研究发展计划(2002AA206653)
关键词
蚓激酶
重组逆转录病毒
包装
转染
表达
lumbrokinase
recombinant retrovirus
package
transfection
expression
