scFv-LAA0融合蛋白的制备及对人肺腺癌细胞H460、小鼠肺腺癌增殖抑制作用观察

摘要

Objective To prepare the fusion protein of recombinant anti-interleukin-4 receptor (IL-4R)single anti-body gene and Trimeresurus stejnegeri venom L-amino acid oxidase (LAAO)gene (scFv-LAAO fusion protein),and then to observed its anti-proliferative effect on human lung adenocarcinoma H460 cells and mouse pulmonary adenocarcinoma cells.Methods ① We connected IL-4R scFv gene and LAAO gene to make the scFv-LAAO fusion gene by splicing o-verlapping extension PCR (SOE-PCR),constructed the pET28a-scFv-LAAO recombinant plasmid,and then used the plas-mid to transfect the BL21(DE3)prokaryotic expression bacterium and induced it to express the scFv-LAAO fusion protein. The molecular weight of scFv-LAAO fusion protein was detected by SDS-PAGE,and the expression of His-tag of scFv-LAAO fusion protein was detected by Western blotting.② Setting the control group,cyclophosphamide group and fusion protein group (including high-dose group,medium-dose group and low-dose group),and in the six groups we separately cultured the human lung adenocarcinoma cells H460 with cell culture medium,cyclophosphamide (2.0 mg/mL),and scFv-LAAO fusion protein (4.0,2.0 and 1.0 mg/mL).We detected and calculated the inhibition rates of cell prolifera-tion of the cyclophosphamide group and fusion protein group (high-dose group,medium-dose group and low-dose group)by MTT.Seventy-five mice were selected to establish the animal models of lung adenocarcinoma,and then were randomly di-vided into the control group,cyclophosphamide group and fusion protein group (including high-dose group,medium-dose group and low-dose group),and the six groups were separately given physiological saline,cyclophosphamide (0.1 mL/10g),scFv-LAAO fusion protein (100,50 and 20 mg/kg)through intraperitoneal injection once per day for 10 days.Sev-en days after drug withdrawal,we weighed the mice of each group,calculated the anti-tumor rate of cyclophosphamide group,high-dose group,medium-dose group and low-dose group of fusion protein,at the same time we recorded the surviv-al time of statistics.Results The length of scFv-LAAO fusion gene was 2 000~3 000 bp.After the prokaryotic expression bacterium BL21(DE3)was transfected by the fusion gene,the fusion protein scFv-LAAO was extracted from the superna-tant of bacteria lysate,and its molecular weight was about 86 kD,which coincided with scFv-LAAO fusion protein,and the His-tag of scFv-LAAO fusion protein was detected by Western bloting.No significant difference was found in the cell prolif-eration inhibition rate,anti-tumor rate and survival time of human lung adenocarcinoma H460 cells between the cyclophos-phamide group and high-dose fusion protein group (all P>0.05 ).The cell proliferation inhibition rate,anti-tumor rate and survival time of human lung adenocarcinoma H460 cells of the cyclophosphamide group and high-dose fusion protein group was higher than that of the medium-dose and low-dose fusion protein groups (all P<0.05 ).Meanwhile,the cell proliferation inhibition rate,anti-tumor rate and survival time of the low-dose group was lower than that of the medium-dose group (all P<0.05).Conclusion The scFv-LAAO fusion protein is successfully prepared,and ScFv-LAAO fusion pro-tein can inhibit the proliferation of human lung adenocarcinoma H460 cells and mouse lung adenocarcinoma cells.%目的：制备重组抗白介素4受体（IL-4R）单链抗体基因与竹叶青蛇毒L-氨基酸氧化酶（LAAO）的融合蛋白（scFv-LAAO融合蛋白），并观察其对人肺腺癌细胞H460、小鼠肺腺癌增殖抑制作用。方法①采用重叠延伸PCR技术将抗IL-4R单链抗体与竹叶青蛇毒LAAO基因连接成scFv-LAAO融合基因，构建重组质粒pET28a-scFv-LAAO，转染BL21（DE3）原核表达菌，诱导表达scFv-LAAO融合蛋白，用SDS-PAGE法检测scFv-LAAO融合蛋白的分子量，用Western blotting法检测scFv-LAAO融合蛋白的His-tag表达。②设空白对照组、环磷酰胺组和融合蛋白组（包括高、中、低剂量融合蛋白组），分别用培养基、2．0 mg／mL环磷酰胺及4．0、2．0、1．0 mg／mL的scFv-LAAO融合蛋白培养人肺腺癌H460细胞，用MTT法测算环磷酰胺组和高、中、低剂量融合蛋白组细胞增殖抑制率。取75只小鼠，建立荷肺腺癌动物模型，随机分为空白对照组、环磷酰胺组和融合蛋白组（包括高、中、低剂量融合蛋白组），分别腹腔注射生理盐水、0．1 mL／10 g体质量的环磷酰胺和100、50和20 mg／kg的scFv-LAAO融合蛋白，1次／d，连用10 d后停药，停药7 d后称量各组小鼠瘤重，计算环磷酰胺组和高、中、低各剂量融合蛋白组抑瘤率；同时统计存活时间。结果 scFv-LAAO融合基因长度2000～3000 bp。用融合基因转染BL21（DE3）原核表达菌后，将细菌裂解从裂解液中分离出的蛋白分子量86 kD，与scFv-LAAO融合蛋白分子量相符，且用Western blotting法在分离到的蛋白中检出scFv-LAAO融合蛋白组氨酸标签。环磷酰胺组和高剂量融合蛋白组人肺腺癌H460细胞增殖抑制率、小鼠抑瘤率和生存时间相比，P均＞0．05；环磷酰胺组和高剂量融合蛋白组H460细胞增殖抑制率、小鼠抑瘤率和生存时间高于低、中剂量融合蛋白组，P均＜0．05；低剂量融合蛋白组H460细胞增殖抑制率、小鼠抑瘤率和生存时间低于中剂量融合蛋白组，P均＜0．05。结论成功制备scFv-LAAO融合蛋白。scFv-LAAO融合蛋白可以抑制人肺腺癌细胞H460、小鼠肺腺癌增殖。