CAJ | 학술논문

目的　获得抗猕猴免疫缺陷病毒(SIV)转基因的淋巴细胞。方法　将锤头型核酶切割的一段靶序列连接在核酶下游构成自切割核酶，人工合成的自切割核酶基因扩增并克隆在pBluescript SK的XhoI-SalI位点，通过连续4次克隆获得10拷贝核酶基因的载体。转染前测定自切割核酶的体外活性并将之转移至哺乳动物表达载体上，脂质体法转染含核酶基因的表达载体，转染细胞在含400 mg/L G418 的培养介质中筛选。转基因细胞对猴免疫缺陷病毒的抗性通过免疫荧光测定来估测。结果　37℃孵育15 min后85％的十体核酶自切割成单体核酶。在初始25 min的反式切割反应中尽管10体核酶摩尔数不足单体核酶十分之一，但十体核酶平均超过单体核酶13.31％的切割产物。Northern 点杂交证明单体核酶基因和十体核酶基因已经转入细胞并在细胞中获得表达。SIV攻毒试验表明，SIV接种6 d后转染单体核酶基因的细胞生长正常，不出现融合细胞，绝大多数转染十体基因的细胞生长良好，只发现极少数的融合细胞；转染单体核酶基因细胞的SIV抗原阳性细胞抑制率达到100％，转染十体核酶基因细胞的SIV抗原阳性细胞抑制率为91.2%, 转空表达载体细胞为48％。结论　转自切割核酶基因的细胞对SIV的感染和致病均表现较强的抗性，但单体核酶比10体核酶对SIV的感染和致病表现出更好的抑制效果。
목적　획득항미후면역결함병독(SIV)전기인적림파세포。방법　장추두형핵매절할적일단파서렬련접재핵매하유구성자절할핵매，인공합성적자절할핵매기인확증병극륭재pBluescript SK적XhoI-SalI위점，통과련속4차극륭획득10고패핵매기인적재체。전염전측정자절할핵매적체외활성병장지전이지포유동물표체재체상，지질체법전염함핵매기인적표체재체，전염세포재함400 mg/L G418 적배양개질중사선。전기인세포대후면역결함병독적항성통과면역형광측정래고측。결과　37℃부육15 min후85％적십체핵매자절할성단체핵매。재초시25 min적반식절할반응중진관10체핵매마이수불족단체핵매십분지일，단십체핵매평균초과단체핵매13.31％적절할산물。Northern 점잡교증명단체핵매기인화십체핵매기인이경전입세포병재세포중획득표체。SIV공독시험표명，SIV접충6 d후전염단체핵매기인적세포생장정상，불출현융합세포，절대다수전염십체기인적세포생장량호，지발현겁소수적융합세포；전염단체핵매기인세포적SIV항원양성세포억제솔체도100％，전염십체핵매기인세포적SIV항원양성세포억제솔위91.2%, 전공표체재체세포위48％。결론　전자절할핵매기인적세포대SIV적감염화치병균표현교강적항성，단단체핵매비10체핵매대SIV적감염화치병표현출경호적억제효과。
Objective　To obtain transgenic lymphocytes that are resistant to the infection and pathogenesis of simian immunodeficincy virus (SIV). Methods　A self-cleaving ribozyme was formed by introducing a fragment of ribozyme target sequence cleft by hammerhead ribozyme to the ribozyme downstream. Self-cleavable ribozyme gene was synthesized, amplified and cloned at the XhoI SalI site of pBluescript SK. The construct bearing 10 copies of ribozyme was obtained through successively cloning four times. The in vitro activity of self-cleavable ribozyme was tested, and then the self-cleaving ribozyme gene was transferred into mammalian expression vector pCI before transfection. The expression vectors harboring self-cleavable ribozyme gene were transfected into lymphocytes with liposome-mediated transfection method. The transfected cell clones were selected in the culturing medium containing 400 mg/L G418. The resistance of transgenic cell to simian immunodeficency virus infection was evaluated by immunofluorescence assays. Results　more than 85% of decameric ribozyme self-cleaved into monomeric ribozymes after incubation for 15 minutes at 37℃. The number of cleavage products of decameric ribozyme during the first 25 minutes exceeds those of the monomeric ribozyme by 13.31% though the molar mass average of decameric ribozyme is less than 1/10 of that of the monomer ribozyme. The Northern dot blot results demonstrated that the monomer ribozyme gene and the decameric ribozyme gene had been transfected and expressed in the cells. SIV challenge trials showed that after 6 days after inoculation of SIV the cells transfected with the monomer ribozyme gene grew normally, no fused cell was observed; and most of the cells transfected with decameric gene grew well, but a few fused cells were observed; The inhibition rate was 100%for the cells transfected with monomer ribozyme gene to the SIV antigen positive cells, 91.2% for the cells transfected with decameric ribozyme gene, and 48% for the cells transfected with the empty expression vector. Conclusion　The lymphocytes transfected with self-cleavable ribozyme gene make a strong resistance to SIV infection and pathogenesis. The cells transfected with monomeric ribozyme gene show a better inhibiting effect on SIV infection and pathogenesis in comparison with the cells transfected with decameric ribozyme gene.