摘要
以ＰＣＲ技术扩增含有ＰｒｅＣ信号肽序列及完整的ＨＢｅＡｇ基因的序列（即ＨＢｃＡｇ基因５′端４４７ｂｐ），在５′端加上合适的酶切位点，克隆到家蚕核多角体病毒转移载体ｐＢｍ０３０上，与野生型ＢｍＮＰＶＤＮＡ共转染家蚕ＢｍＮ细胞，空斑纯化后得到多角体基因失活的重组病毒。ＥＬＩＳＡ法测定表明培养液上清中ＨＢｅＡｇ效价达１∶３２０００，细胞内ＨＢｅＡｇ效价为１∶２０００，培养液及细胞内的ＨＢｃＡｇ含量极低（＜１∶１６０）。研究结果表明，ＢｍＮ细胞能正确识别与切割ＨＢｅＡｇ信号肽序列，所表达的ＨＢｅＡｇ效价高，纯度好。
A HBeAg gene fragment, which has some restriction endonucleased sites on both 5′ ends, including pre c signal peptide sequence and 5′ 447 bp of the HBcAg gene was amplified by PCR. The HBeAg gene fragment was cloned into the BmNPV transfer vector pBm030 and the chimeric vector pBmHBe was constructed. The BmN cells were co transfected with pBmHBe and Wt BmNPV DNA,therefore, the recombinant viruses were obtained by plaque purification. Analysis of the HBeAg antigenecity by ELISA showed that the highest titer in the cell cultural medium was up to a dilution of 1∶32000. Although HBeAg protein also presents in the BmN cells the titer was only 1:2000. The HBcAg protein was fewer than HBeAg (<1∶160) whatever in culture medium and in cells. the results showed that the BmN cells can recognize the HBeAg signal peptide sequence and cut it correctly for HBeAg. The BmN PV BmN cell system is considered to be much better than E.coli system for producing HBeAg protein.
出处
《中国病毒学》
CSCD
1998年第1期70-76,共7页
Virologica Sinica
基金
江苏省自然科学基金
