【作者】 吕建新；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 目的将人白介素-18（human interleukin 18,hIL18）基因与表皮生长因子受体干扰序列（EGF loop C sequence,EGF）构建IL18-EGF融合基因,利用pET32a/E.coli BL21（DE3）表达IL18-EGF融合基因,通过复性、纯化获得有活性的重组人IL18-EGF融合蛋白,通过体内、体外试验评价IL18-EGF融合蛋白的抗肿瘤活性。方法1.hIL18 cDNA克隆与rhIL18-EGF融合基因的构建:从1名健康献血者的外周血单个核细胞（PBMC）中提取总RNA,RT-PCR扩增hIL18 cDNA,通过PCR重组技术,将hIL18与EGF序列通过一个中间接头（Gly4Ser）3编码序列融合,IL18-EGF融合基因与pMD18-T载体连接,命名为pFUS-IL18-EGF质粒,并测序鉴定。2.IL18-EGF融合蛋白原核表达载体的构建和表达:采用PCR方法,以质粒pFUS-IL18-EGF为模板,扩增IL18-EGF融合基因,将其克隆入原核表达质粒pET32a（+）,构建表达载体pET32a-IL18-EGF,限制性内切酶双酶切和测序鉴定质粒构建是否正确。将表达质粒转化E.coli Rosetta（DE3）,IPTG诱导其表达,SDS-PAGE和免疫印迹分析表达产物。3.IL18-EGF融合蛋白包涵体的复性、纯化:大量表达分离IL18-EGF融合蛋白包涵体,并用2 M尿素和1%Triton X-100反复洗涤。以8M尿素溶解包涵体,将包涵体裂解液上样于弱阴离子交换柱DEAE-52,进行柱上复柱,复性产物进一步用分子筛Sephadex G-75纯化。复性纯化产物与人外周血单个核细胞（PBMC）共培养24h,RT-PCR检测PBMC IFN-γmRNA的表达情况来评价复性效果。4.IL18-EGF融合蛋白的体内抗肿瘤活性研究:皮下注射2×10⁶人肝癌细胞株SMMC-7721细胞至Balb/c裸鼠右侧腰部;成瘤后,将裸鼠进行随机化分成IL18-EGF组、rhIL18组和生理盐水（NS）空白对照组;待肿瘤直径为0.5～0.7cm时,瘤周注射1×10⁶经8Gy放射线处理的NK-92细胞,各组裸鼠再用相应的药物瘤周注射进行治疗,每5天注射NK细胞和药物一次,共持续5次。在治疗开始之前及在各次注射治疗的时间点均测量各移植肿瘤的长短径,计算肿瘤体积。同时观察并记录裸鼠摄食、饮水等其他生长情况。在最后一次治疗的次日,通过拉颈处死全部裸鼠,切除肿瘤组织。一部分肿瘤组织制成单细胞悬液并应用流式细胞术进行细胞周期和凋亡的监测;另一部分肿瘤组织,连同裸鼠的肝、肺、脾等器官一并通过10%福尔马林的固定用于常规病理切片染色和观察。5.IL18-EGF融合蛋白的促增殖、诱导分泌IFN-γ和活化免疫细胞的体外研究:从健康志愿者抽取静脉血5ml,密度梯度离心分离外周血单个核细胞（PBMNCs）。利用CCK-8试剂盒测试IL18-EGF对PBMNCs的促增殖作用。应用ELISA法检测IL18-EGF诱导KG-1细胞分泌IFN-γ。应用多参数流式细胞术检测IL18-EGF对SMMC-7721肿瘤抗原和K562细胞抗原致敏的PBMNCs淋巴细胞亚群分布及CD4+和CD8+T细胞活化的影响。6.IL18-EGF融合蛋白体内、体外试验的统计学分析:应用SPSS13.0统计软件包、Prism 4.0统计软件进行数据分析,CurveExpert 1.3免费软件用于标准曲线的制作。结果1.突变型IL18 cDNA的克隆:RT-PCR扩增出人IL18 cDNA,PCR产物经限制性核酸内切酶酶切分析以及测序结果Blast同源性分析证明,该序列与Ushio等报道的序列相比存在G157A（Asp→Asn）、G364A（Met→Val）、G484A（Ala→Thr）等3处有意义突变,属于突变型,GenBank收录（AF380360）。2.PCR法构建IL18-EGF融合基因:以IL18-EGF融合基因与pMD18-T载体连接,该质粒命名为pFUS-IL18-EGF质粒。pFUS质粒经SalⅠ/XbaⅠ双酶切和测序验证,IL18-EGF融合基因与原设计的基因序列相符合,GenBank收录（AF454397）。经计算机模拟分析,IL18-EGF融合蛋白与IL18自然成熟胎在分子疏水性、二级结构等理化特性方面相似。3.IL18-EGF融合蛋白原核表达载体成功构建和表达:双酶切分析以及测序结果表明原核表达载体pET32a-IL18-EGF与设计相符;工程菌E.coli Rosetta（DE3）/pET32a-IL18-EGF诱导表达后在分子量为37kDa处可产生特异的表达条带,并能够被抗人IL18单克隆抗体所识别,与预期一致。4.IL18-EGF融合蛋白包涵体的复性、纯化及其生物学活性测定:变性的IL18-EGF融合蛋白包涵体经阴离子交换层析柱DEAE-52柱上复性和分子筛Sephadex G-75进一步纯化后纯度达95%。RT-PCR检测结果表明复性产物具有诱导人PBMC IFN-γ基因表达的能力。IFN-γELISA结果显示,IL18-EGF融合蛋白具有明显地诱导KG-1细胞产生IFN-γ的能力,但活性低于标准rhIL18。5.裸鼠体内IL18-EGF融合蛋白抗肿瘤试验中,瘤周注射IL18-EGF合并NK细胞注射能显著抑制SMMC-7721移植瘤的生长:在治疗结束时,SMMC-7721异种移植瘤的体积在IL18-EGF组、rhIL18组和空白对照组分别为0.063±0.030cm³,0.212±0.037cm³和0.432±0.030cm³空白对照组与IL18-EGF组和rhIL18组的差别是比较明显的（P＜0.01）。此外,rhIL18组与IL18-EGF组的差别也具有统计学意义（P=0.05）。细胞周期分析表明,IL18-EGF组有96.11%±1.51%的细胞停滞在G1期,而在rhIL18组和空白对照组则分别是81.3%±2.02%和72.18%±0.46%,前者与后两者相比均有明显的差异（IL18-EGF vs rhIL18:P＜0.05;IL18-EGF vs control:P＜0.01）。同时,rhIL18组与空白对照组相比也有明显差异（P＜0.05）。IL18-EGF组、rhIL18组和空白对照组S期细胞的比例分别为3.69%±0.41%,16.51%±1.25%,和23.85%±1.36%,前者与后两者相比均有明显的差异（IL18-EGF vs rhIL18:P＜0.01;IL18-EGF vs control:P＜0.01）。同时,rhIL18组与空白对照组相比也有明显差异（P＜0.05）。肿瘤细胞自主凋亡发生率在三组中也有明显不同,与空白对照组（7.10%±1.13%）相比,IL18-EGF组（28.05%±3.89%,P＜0.05）、rhIL18组（25.48%±5.46%,P＜0.05）肿瘤细胞自主凋亡比例明显增高,而IL18-EGF组和rhIL18组间无明显差异。肿瘤标本切片观察未发现各治疗组病理特征有明显差异,在各主要脏器中均未发现肿瘤转移。此外,各治疗组裸鼠在体重、摄食及饮水等方面也无明显改变。6.IL18-EGF融合蛋白的促增殖、诱导分泌IFN-γ的体外研究:PBMNCs在IL18-EGF组、rhIL18组和空白对照组中的增殖活性(OD₄₅₀)前两者与空白对照组相比均有明显差异（IL18-EGF vscontrol:P＜0.01;IL18 vs control:P＜0.05）,分别为1.14±0.07,1.02±0.42和0.84±0.01,而IL18-EGF组和rhIL18组间无明显差异。IL18-EGF能有效诱导KG-1细胞合成、分泌IFN-γ。IL18-EGF组、rhIL18组和空白组培养上清中IFN-γ的水平分别为96.67pg/ml、164.0pg/ml及12.33pg/ml。7.IL18-EGF融合蛋白活化免疫细胞的体外研究:PBMNCs在更多还原

【Abstract】 ObjectiveTo construct IL18-EGF fusion gene harboring human epidermal growth factor receptor interfere sequence(EGF loop C sequence,EGF) and human Interleukin-18(hIL18),to express the IL18-EGF fusion gene in pET32/E.coli BL21(DE3),and to evaluate antineoplastic bioactivities of IL18-EGF fusion protein in vivo and in vitro.Methods1:Construction of rhIL18-EGF fused gene:Total RNA was extracted from Peripheral Blood Mononuclear Cells(PBMC).After amplification of the hIL18 gene by RT-PCR,we linked it with EGF gene and constructed pFUS-IL18-EGF by gene recombination.2:Expression and identification of human IL18-EGF fused gene: Prokaryotic expression vector pET32a-IL18-EGF was constructed by recombinant DNA technique and later identified by BamHⅠ/Bpull02Ⅰdouble digestion and gene sequencing.Then recombinant vector was further transformed into E.coli Rosetta(DE3)for expression under the induction of IPTG at 25℃.SDS-PAGE and Western blot were applied to confirmation of expression product.3:Purification and renaturation of the fusion protein:The inclusion body of the fusion protein was washed with 2 M carbamide and 1% Triton X-100 followed dissolving in 8 M carbamide,lysate of inclusion body was renatured by DEAE-52 anion exchange column.Fusion protein was co-cultured with(PBMC)for 24h,then expression levels of mRNA in PBMC IFN-γwas measured by real time RT-PCR.4:Antineoplastic activity of the IL18-EGF fusion protein in vivo: 2×10~6 human hepatic carcinoma cell lines SMMC-7721 cells cultured in DMEM(high glucose)supplemented with 10%fetal bovine serum were subcutaneously injected into the right flank of Balb/c nude mice.Balb/c nude mice with established xenograft were randomized into three groups of treatment:IL18-EGF,rhIL18 and blank group.Tumor-bearing mice were treated with peritumoral subcutaneously injection of 1×10~6 NK-92 cells irradiated with 8Gy X-ray followed by peritumoral subcutaneously injection of three groups,every five days for five times.The next morning after the last dosage,all mice were sacrificed through cervical dislocation and the tumor masses and mouse organs(liver,spleen and lung)were completely dissected for further usage.5:Abilities to promote proliferation,induce to secrete IFN-γand active immune cells:In vitro experiments,peripheral blood mononucleated cells(PBMNCs)were isolated with gradient centrifugation and the effects of IL18-EGF on proliferation of PBMNCs were measured by CCK-8 kit.IFN-γproduction by KG-1 cells was assayed with ELISA kit.Flow cytometry with the built-in software packages was carried out to observe how IL18-EGF actived SMMC-7721 neoantigen,the distributions of lymphocyte subpopulations sensitized by antigens of K562 cells,and the activation of CD4+and CD8+T lymphocytes..6:Statistic analysis:SPSS 13.0 software package,Prism 4.0 software package and Curve Expert 1.3 free software were used for statistic analysis,and P＜0.05 was considered significant throughout in this work. Results1:Clone of mutant IL18 cDNA:Amplification of IL18 gene by RT-PCR, restriction enzyme zymogram analysis,PCR,DNA sequencing and BLAST search indicated that there were 3 mutations compared with sequences reported by Ushio,including G157A(Asp→Asn),G364A (Met→Val),G484A(Ala→Thr),which have been accepted by GenBank (AF380360).2:Construction of IL18-EGF fused gene by PCR:Two-step PCR products of the IL18-EGF fused gene were cloned into pMD18-T vector to construct pFUS-IL18-EGF.Then the plasmid pFUS was confirmed by SalⅠ/XbaⅠdouble digestion and sequencing,accepted by GenBank. (AF454397).Computer simulation analysis showed that physicochemical properties like hydrophobicity and secondary structure of IL18 structural domain of IL18-EGF fusion protein were familiar with that of the natural IL18 structural domain.3:Prokaryotic expression vector was constructed and fusion protein was expressed successfully:Double digestion and sequencing confirmed the previous design of the prokaryotic expression vector,and expressed fusion protein with 37kDa was effectively identified by IL18 monoclonal antibody.4:Purification and renaturation of the IL18-EGF fusion protein and measurement of its activity:Purity of fusion protein reached 95%after purification and renaturation by DEAE-52 anion exchange column and Sephadex G-75 molecular sieve.RT-PCR indicated that purified product could induce the expression of the PBMC IFN-γgene.Moreover,IFN-γELISA tests showed fusion protein had effectively induced KG-1 cells to secret IFN-γ,even though the ability was lower than the standard IL18.5:Subcutaneous injection of IL18-EGF and NK cells adjacent to tumor significantly inhibited the growth of SMMC-7721transplantation tumor:Before the end of therapy,we found volume of xenograft tumor was(0.063±0.030)mm3 in IL18-EGF group,(0.212±0.037)mm3 in rhIL18 group,and(0.432±0.030)mm3 in control group respectively.Compared to control,SMMC-7721 xenograft growth in Balb/c nude mice was markedly inhibited with peritumoral injection of IL18-EGF(P＜0.01).Furthermore,the inhibitory effect of IL18-EGF on SMMC-7721 growth in vivo was more profound than that of rhIL18(P=0.05).Meanwhile,more tumor cells were arrested in G1 phase in IL18-EGF group(96.11±1.51%),compared to rhIL18 group (81.3±2.02%,P＜0.05)as well as to control(72.18±0.46%,P＜0.01).The difference on the percentage of G1 phase between rhIL18 group and control was also significant(P＜0.05).The percentages of S phase in IL18-EGF group,rhIL18 group and control were 3.69±0.41%, 16.51±1.25%,and 23.85±1.36%,respectively(IL18-EGF vs rhIL18:P＜0.01;IL18-EGF vs control:P＜0.01;IL18 vs control:P＜0.05).Tumor spontaneous apoptotic rates in IL18-EGF,rhIL18 and medium group were 28.05±3.89%(P＜0.05,compared to control),25.48±5.46%(P＜0.05,compared to control)and 7.10±1.13%,respectively.And there was still not significant difference between IL18-EGF and rhIL18 group on spontaneous apoptosis induction.No significant difference was observed on pathologic features of SMMC-7721 xenograft grown in Balb/c nude mice treated with IL18-EGF,rhIL18 and control,respectively.In addition, no apparent unwanted reaction was detected in mice treated with IL18-EGF as demonstrated in mice weight comparison undertaken at the end of treatment.6:IL18-EGF fusion protein to promote proliferation and induce to secrete IFN-γin vitro:The proliferation of PBMNCs induced by IL18-EGF,rhIL18 and medium in the presence of 0.5ug/ml ConA were 1.14±0.07,1.02±0.42 and 0.84±0.01,respectively,indicating significant pro-proliferation activity of IL18-EGF(P＜0.01)and of rhIL18(P＜0.05) as compared to control.However,there was not significant difference between IL18-EGF and rhIL18 on proliferation of PBMNCs.IFN-γ更多还原