CAJ | 학술논문

目的构建HIV-1 CN54株gagprotease基因嵌合脊髓灰质炎病毒cDNA的表达质粒,并鉴定、检测基因及其表达.方法用PCR技术获得人免疫缺陷病毒CN54株的gagprotease基因,并使其两端带上合适的酶切位点,将其定向插入到包含脊髓灰质炎病毒cDNA的表达质粒pSVA14中,替代其部分结构基因,构建HIV基因嵌合缺陷性脊髓灰质炎病毒基因组的表达质粒.经筛选、鉴定后用脂质体转染技术将新构建的质粒转入Hela细胞内,用Western Blot方法检测目的基因在Hela细胞内的表达.结果 PCR技术扩增所得的人免疫缺陷病毒CN54株gagprotease基因经琼脂糖凝胶电泳、DNA测序证实成功获得,未引入突变碱基,筛选、鉴定证明gagprotease基因被正确定向插入到脊髓灰质炎病毒的cDNA序列之中,Western Blot检测到gagprotease基因正确表达了相关蛋白.结论成功构建了表达人免疫缺陷病毒CN54株gagprotease基因的缺陷性脊髓灰质炎病毒基因组嵌合质粒,为利用脊髓灰质炎病毒作人免疫缺陷病毒基因的表达载体奠定了基础,此研究对开发以脊髓灰质炎病毒为艾滋病的疫苗载体有重要意义.
목적 구건HIV-1 CN54주gagprotease기인감합척수회질염병독cDNA적표체질립,병감정、검측기인급기표체.방법 용PCR기술획득인면역결함병독CN54주적gagprotease기인,병사기량단대상합괄적매절위점,장기정향삽입도포함척수회질염병독cDNA적표체질립pSVA14중,체대기부분결구기인,구건HIV기인감합결함성척수회질염병독기인조적표체질립.경사선、감정후용지질체전염기술장신구건적질립전입Hela세포내,용Western Blot방법검측목적기인재Hela세포내적표체.결과 PCR기술확증소득적인면역결함병독CN54주gagprotease기인경경지당응효전영、DNA측서증실성공획득,미인입돌변감기,사선、감정증명gagprotease기인피정학정향삽입도척수회질염병독적cDNA서렬지중,Western Blot검측도gagprotease기인정학표체료상관단백.결론 성공구건료표체인면역결함병독CN54주gagprotease기인적결함성척수회질염병독기인조감합질립,위이용척수회질염병독작인면역결함병독기인적표체재체전정료기출,차연구대개발이척수회질염병독위애자병적역묘재체유중요의의.
[Objective] To construct a chimeric expression plasmid which contains HIV-1 CN54 strain gagprotease gene and Poliovirus cDNA, identify and examine the recombinant plasmid and its gene expression. [Methods]HIV-1 CN54 strain gagprotease gene with cleavage enzyme site in its two ends was obtained through PCR technique. It was orientedly inserted into the expression plasmid pSVA14, replacing one structural gene fragment of Polioviurs. Later the recombinant plasmid was proved correct construction by restriction enzyme cleavage identification.With liposome transfection way, the recombinant plasmid was trasnfected into cultured Hela cells. Western blot was adopted to examine the gene expression. [Results] Proved through electrophoresis in gel, HIV-1 gagprotease gene was successfully amplified. No mutation occurred in its bases identified by gene sequencing. Identification by restriction endonuclease enzyme showed gagprotease gene was correctly inserted into poliovirus cDNA. Western blot test showed that HIV related protein was expressed in cultured Hela cells. [Conclusion] A chimeric Polioviurs-HIV gagproteae gene expression plasmid was constructed, which could provide the base for Poliovirus as a HIV gene expression vector. It is of great significance to invent an AIDS vaccine based on Poliovirus vector.