基于狂犬病病毒G蛋白scFv介导的靶向shRNA制备与鉴定

Targeted inhibition of Rabies Virus replication in vitro by single chain antibody domain mediated vector expression shRNA delivery

【目的】探讨以狂犬病病毒G糖蛋白单链抗体介导的载体表达shRNA靶向制剂,靶向抑制狂犬病毒复制的可行性。【方法】应用PCR技术获得狂犬病毒G糖蛋白单链抗体scFv(G)和绿脓杆菌跨膜区-酵母DNA结合结构域ETA-GAL4基因,通过搭桥PCR法获得scFv(G)-ETA-GAL4(SEG)嵌合基因;克隆至原核表达载体pET28a(+),构建重组表达质粒pET28a(+)-scFv(G)-ETA-GAL4(pET28a-SEG);在大肠杆菌BL21(DE3)中经IPTG诱导表达,利用镍柱亲和层析法纯化包涵体,经复性、鉴定制得SEG蛋白;ELISA法检测表达蛋白与狂犬病毒特异结合活性;将SEG蛋白与含shRNA的质粒(pRNATU6.3-shRNA)连接制成靶向shRNA,接入100 TCID50狂犬病毒感染BHK-21细胞,35 h观察细胞中绿色荧光蛋白(GFP)表达情况;48 h用直接免疫荧光抗体试验测定复合物抑制病毒效果。【结果】克隆得到1557 bp的SEG蛋白编码基因,大肠杆菌中成功表达57 KDa的SEG蛋白,能与抗His的单克隆抗体发生特异性反应,SEG蛋白经镍柱纯化、复性后得率为2.8 mg/mL。ELISA试验证明SEG蛋白在一定浓度范围内与RV结合呈正相关。细胞试验表明GFP在细胞内得到表达;直接免疫荧光试验测定该复合物能抑制76%病毒复制。【结论】SEG蛋白能与携带shRNA的质粒结合,可运送该质粒至RV感染BHK-21细胞中,抑制狂犬病毒的复制。

[ Objective] Single chain antibody-mediated delivery is a novel approach for targeting shRNA to appropriate cells. In this report, we studied whether this shRNA delivery strategy would be effective against rabies virus. [ Methods] Rabies virus scFv （G） gene and ETA-GAIA gene were amplified by PCR from vector seFv （G）-T and PE40-GAL4-T respectively. Then, the chimeric gene scFv（G）-ETA-GAL4 was constructed by lapextension PCR and cloned into the prokaryotie expression vector pET28a（ ＋ ）. Recombinant expression plasmid of pET28a （ ＋ ）-seFv （G）-ETA-GAIA was constructed and then transformed into the competent E. coli BL21 （ DE3 ） cells for expression under the induction of IPTG. scFv（G） -ETA-GAIA protein was purified by Ni-NTA His Bind Resin affinity chromatograph and identified by SDS-PAGE gel and Western blot assay. Binding of the fusion protein scFv（G）-ETA-GAL4 to rabies virus was determined by ELISA. Complexes which formed spontaneously by the fusion protein scFv （G）-ETA-GAL4 with plasmid pRNATU6.3-shRNA were added to BHK-21 cells culture medium that infected with RV. Green fluorescent protein （GFP） was observed after 35 h and judged the transferring efficiency of the complexes. The inhibition of RV replication by shRNA was detected by direct immune fluorescence test. [Results] A 1557 bp DNA encoding scFv（ G）-ETA-GAIA protein gene was cloned and successfully expressed in inclusion body with approximate molecular weight of 57.0 kDa, which could be recognized by anti-His mAb. The seFv（G）-ETA-GAlA proteins were purified by Ni-NTA column , and after renatured with the yield of 2.8 mg/mL. The ELISA results showed that when concentration of the scFv （G）-ETA-GAL4 protein ranging from 2.8 nmol/L to 1000 nmol/L, binding affinity is directly related with RV. The GFP expressed in BHK-21 cell after transfection with the complexes and effectively inhibited RV replication in BHK-21 cell. [ Conclusion ] scFv （G）-ETA- GAIA fusion protein could mediated plasmid pRNATU6.3-shRNA transferred into BHK-21 cell infected with RV, and then inhibited RV replication.