CAJ | 학술논문

目的合成和筛选nogc66-siRNA干扰片段,克隆神经生长因子-β(NGF-β),构建含nogu66-siRNA和NGF-β双基因的重组腺相关病毒穿梭质粒.方法设计针对nogo66的siRNA,插入载体pGenesil-1.1,转染大鼠胶质瘤C6细胞,通过RT-PCR和Western-blot检测筛选有效的干扰质粒;培养人MI-Apaca-2细胞,提取总RNA,进行RT-PCR得到人NGF-β基因,插入T-easy载体;将pGenesil-1.1与pAAV-MCS进行重组,得到含U6和CMV双启动子的重组腺相关病毒穿梭质粒pAAV-U6/CMV-EGFP;将nogo66-siRNA和NGF-β分别插入pAAV-U6/CMV-EGFP中,构建含nogo66-siRNA和NGF-β双基因的重组腺相关病毒穿梭质粒.结果干扰质粒pGenesil-nogo66-siRNA测序显示干扰序列正确;RT-PCR结果显示转染后各条带相对亮度有差异,转染pGenesil-nogo66-siRNA-1和pGenesil-nogo66-siRNA-2使nogo基因表达降低;Western-blot结果显示转染pGenesil-nogo66-siRNA-1使NOGO蛋白表达下降73%,转染pGenesil-nogo66-siRNA-2使NOGO蛋白表达下降78%,与未转染组相比,差异有统计学意义(P<0.05);质粒T-easy-NGF-β酶切可见3000 bp、736 bp条带,测序显示基因序列正确;酶切pAAV-U6/CMV-EGFP、pAAV-U6-nogo66-siRNA/CMV-EGFP可见4300 bp、800 bp条带,转染细胞后均可见绿色荧光蛋白表达;酶切pAAV-U6/CMV-NGF-β、pAAV-U6-nogo66-siRNA/CMV-NGF-β可见4300 bp、736 bp条带;质粒pAAV-U6βnogo66-siRNA/CMV-NGF-β测序显示载体中的基因序列止确.结论成功构建了含有nogo66-siRNA与NGF-β双基因的重组腺相关病毒穿梭质粒,为深入研究脊髓损伤的修复提供了实验基础.
목적 합성화사선nogc66-siRNA간우편단,극륭신경생장인자-β(NGF-β),구건함nogu66-siRNA화NGF-β쌍기인적중조선상관병독천사질립.방법 설계침대nogo66적siRNA,삽입재체pGenesil-1.1,전염대서효질류C6세포,통과RT-PCR화Western-blot검측사선유효적간우질립;배양인MI-Apaca-2세포,제취총RNA,진행RT-PCR득도인NGF-β기인,삽입T-easy재체;장pGenesil-1.1여pAAV-MCS진행중조,득도함U6화CMV쌍계동자적중조선상관병독천사질립pAAV-U6/CMV-EGFP;장nogo66-siRNA화NGF-β분별삽입pAAV-U6/CMV-EGFP중,구건함nogo66-siRNA화NGF-β쌍기인적중조선상관병독천사질립.결과 간우질립pGenesil-nogo66-siRNA측서현시간우서렬정학;RT-PCR결과현시전염후각조대상대량도유차이,전염pGenesil-nogo66-siRNA-1화pGenesil-nogo66-siRNA-2사nogo기인표체강저;Western-blot결과현시전염pGenesil-nogo66-siRNA-1사NOGO단백표체하강73%,전염pGenesil-nogo66-siRNA-2사NOGO단백표체하강78%,여미전염조상비,차이유통계학의의(P<0.05);질립T-easy-NGF-β매절가견3000 bp、736 bp조대,측서현시기인서렬정학;매절pAAV-U6/CMV-EGFP、pAAV-U6-nogo66-siRNA/CMV-EGFP가견4300 bp、800 bp조대,전염세포후균가견록색형광단백표체;매절pAAV-U6/CMV-NGF-β、pAAV-U6-nogo66-siRNA/CMV-NGF-β가견4300 bp、736 bp조대;질립pAAV-U6βnogo66-siRNA/CMV-NGF-β측서현시재체중적기인서렬지학.결론 성공구건료함유nogo66-siRNA여NGF-β쌍기인적중조선상관병독천사질립,위심입연구척수손상적수복제공료실험기출.
Objective To construct a recombinant adeno-associated virus shuttle vector co-expressing nogo66-siRNA and NGF-βgenes after nogo66-siRNA is synthesized and screened to clone NGF-βgene. Methods After the nogo66-siRNA fragments were designed and cloned into pGenesil-1. 1,they were transfected into C6 cell line. RT-PCR and Western-blot were used to detect the expression of nogo gene and select the plasmid of effective interference. T-easy-NGF-βvector was constructed by cloning human NGF-βgene into T-easy vector and identified by PCR, digestion and DNA sequencing. The pAAV-U6/CMV-EGFP containing U6 promoter and CMV promoter was constructed by restructuring pGenesil-1. 1 and pAAV-MCS. As nogo66-siRNA and NGF-βgenes were separately cloned into the pAAV-U6/CMV-EGFP vector,the recombinant adeno-associated virus shuttle vector co-expressing nogo66-siRNA and NGF-βgenes was obtained and identified by PCR, digestion and DNA sequencing. Results DNA sequencing results showed correct interference sequences. The bands of 4250 bp and 800 bp were detected when pGenesil-nogo66-siRNAs were digested. RT-PCR and Western-blot results showed that the transfection of pGenesil-nogo66-siRNA-2 made the expression of NOGO protein decline by 78% compared with the non-transfected cells. The bands of 3000 bp and 736 kb were detected when T-easy-NGF-βwas digested. DNA sequencing results of T-easy-NGF-βwere fully consistent The bands of 4250 bp and 800 bp were detected when pAAV-U6/CMV-EGFP and pAAV-U6-nogo66-siRNA/CMV-ECFP were digested. The bands of 3000 bp and 736 kb were detected when pAAV-U6/CMV-NGF-βand pAAV-U6-nogo66-siRNA/CMV-NGF-βwere digested. The green fluorescent protein was detected when pAAV-U6/CMV-EGFP and pAAV-U6-nogo66-siRNA/CMV-EGFP were transfected into C6 cells. DNA sequencing results of pAAV-U6-nogo66-siRNA/CMV-NGF-βshowed that sequences were completely correct Conclusion The adeno-associated virus shuttle vector pAAV-U6-nogo66-siRNA/CM V-NGF-βcan be successfully constructed, providing an experimental basis for further study of repair of spinal cord injury.