【作者】 刘一尘；

【导师】 程安春； 张春杰；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2008， 博士

【摘要】 新城疫病毒(Newcastle disease virus,NDV)是一种溶癌病毒,可在肿瘤细胞内增殖、抑制并裂解肿瘤细胞。研究发现,NDV的包膜糖蛋白血凝素-神经氨酸酶(hemagglutinin-neuraminidase,HN)基因是NDV抗肿瘤的功能性基因,是NDV抗肿瘤作用的重要分子基础。IL-18具有明显的抗肿瘤作用,在肿瘤治疗和肿瘤基因治疗方面具有潜在的应用前景。为探讨HN基因及IL-18基因融合后抗肿瘤作用及其机制,本研究分别构建了HN基因、ChMIL-18基因及HN基因和ChMIL-18基因融合后的重组表达载体pPICZαA-HN、pPICZαA-ChMIL18和pPICZαA-HN-ChMIL18,经诱导表达后,其目的蛋白均具生物学活性,进而以鸡马立克病肿瘤细胞系-MSB-1为体外研究对象,对MSB-1细胞的增殖抑制作用及其机制等方面进行了初步研究,结果表明,HN蛋白、HN-ChMIL18融合蛋白均可抑制肿瘤细胞生长,HN-ChMIL18融合蛋白对细胞抑制作用明显强与HN蛋白,其原因可能是ChMIL-18蛋白对HN蛋白致肿瘤细胞凋亡有增强作用,而ChMIL-18蛋白虽具有淋巴细胞增殖活性,但对肿瘤细胞无明显的抑制作用,其为进一步研究HN基因和ChMIL-18基因融合蛋白体内抗马立克肿瘤奠定了基础,具有重要的理论和实际意义。1目的基因的获得及生物信息学分析1.1 ChMIL-18目的基因的获得根据Genbank中鸡IL-18成熟蛋白基因序列设计一对特异性引物,利用RT-PCR技术,从外周血淋巴细胞中扩增到了鸡IL-18成熟蛋白基因全序列,将其克隆至pMD18-T载体并测序,对测序结果进行生物信息学分析,结果表明:序列全长为507bp,其理论分子量为19.5KD,等电点为6.36,无糖基化位点,具抗原性等。为提高具有生物活性的ChIL-18的表达量,应用反向长距离PCR定点突变技术对鸡IL-18成熟蛋白基因第28位和第37位的Arg密码子进行同义突变,将毕赤酵母中低频密码子CGA突变为高频密码子AGA,获得突变体pMD18-T-ChMIL18,将其转化至E.coli JM109中,抽提质粒,经酶切和PCR鉴定后,进行测序,测序结果与原ChIL18比对表明突变成功,获得了目的突变基因。1.2 HN基因的获得应用RT-PCR方法对NDV LaSota株HN基因进行了扩增与克隆,得到大小约为1.7Kb的LaSota株HN基因,与Genbank中登录的HN基因大小一致:扩增序列经PCR、酶切鉴定及序列测定证实与预计结果相符:用DNAStar及在线分析软件对HN基因的生物信息学进行分析,其结果为:HN基因共577个氨基酸,理论分子量为63.0KD,等电点为7.54,12个半胱氨酸残基,5个N-糖基化位点,与B1、Clone30、LaSota、JI1和HB92核苷酸同源性在98.8%-99.8%,氨基酸同源性分别为98.8%-99.5%。1.3 HN-ChMIL18融合基因的获得根据表达载体pPICZαA及克隆载体pBluescript SK+(pBS SK+)的多克隆位点,设计HN基因的引物(含柔性接头),在两端分别引入KpnI和BamHI酶切位点,以及变构体MIL-18引物(含柔性接头),在两端分别引入BamHI和NotI酶切位点。分别以pMD18-T-HN、pMD18-T-ChMIL18为模板扩增得到融合基因片段HN~f和ChMIL-18~f,并克隆至pMD18-T载体,构建了克隆载体pMD18-T-HN~f、pMD18-T-ChMIL18~f。重组质粒pMD18-T-HN~f和pBS SK+分别用/KpnI/BamHI双酶切,片段回收产物按适当比例连接,转化E.coli JM109感受态细胞,重组质粒命名为pBS SK+-HN。然后,重组质粒pMD18-T-ChMIL18~f和pBS SK+-HN分别用BamHI、NotI双酶切,回收产物按适当比例连接,转化E.coli JM109感受态细胞,重组质粒鉴定正确后命名为pBS SK+-HN-ChMIL18,经酶切、PCR鉴定及序列比对表明获得了目的基因HN-ChMIL18。2表达载体的构建、诱导表达及表达产物的活性检测2.1表达载体pPICZαA-ChMIL18的构建、诱导表达及表达产物的活性检测质粒pMD18-T-ChMIL18和pPICZαA分别用EcoRl、KpnI双酶切,电泳回收纯化ChMIL18和pPICZαA双酶切产物,按适当比例进行连接,转化到E.coli JM109感受态细胞中,鉴定结果表明获得了重组质粒pPICZαA-ChMIL18。将重组质粒pPICZαA-ChMIL18用SacI酶切线性化,电脉冲方法克隆至Pichia Pastoris(P.Pastoris)X-33感受态细胞中,经高抗性筛选、PCR鉴定结果表明pPICZαA-ChMIL18已整合入P.Pastoris X-33染色体中,用1%甲醇对其进行诱导表达,SDS-PAGE结果表明目的蛋白存在于上清中,分子量约为23KD,Western blot结果表明表达产物具良好的免疫原性,淋巴细胞转化试验结果表明ChMIL18具有促淋巴细胞增殖的活性。2.2表达载体pPICZαA—HN的构建、诱导表达及表达产物的活性检测质粒pPICZαA和重组质粒pMD18-T-HN分别用KpnI/NotI双酶切,电泳回收纯化HN和pPICZαA片段,按适当的比例进行连接,克隆至E.coli JM109感受态细胞中,经酶切及PCR鉴定,结果表明获得了重组质粒pPICZαA-HN。重组质粒pPICZαA-HN用SacI酶切线性化,电击转化入P.Pastoris X-33感受态细胞中,经高抗性筛选、PCR鉴定证明pPICZαA-HN已整合入X-33的染色体上,对其进行诱导表达,SDS-PAGE、Western-blot及血凝试验结果表明上清中有目的蛋白,其分子量约为97KD,且具良好的免疫原性和凝集红细胞的活性。2.3表达载体pPICZαA-HN-ChMIL18的构建、诱导表达及表达产物的活性检测质粒pBS SK+ -HN-ChMIL18和pPICZαA分别用KpnI/NotI双酶切,电泳回收HN-ChMIL18与pPICZαA双酶切产物并纯化,按适当比例进行连接,克隆至E.coliJM109感受态细胞,酶切及PCR鉴定结果表明获得了重组质粒pPICZαA-HN-ChMIL18。重组质粒pPICZαA-HN-ChMIL18用SacI酶切线性化后,电击转化入酵母菌X-33感受态细胞中,高抗性筛选和PCR鉴定结果表明pPICZαA-HN-ChMIL18已整合入X-33染色体上,对其进行诱导表达,SDS-PAGE、Western-blot结果表明目的蛋白为分泌性表达,分子量约为103KD,具良好的免疫原性,同时,淋巴细胞转化试验、血凝试验证明表达产物具有促进淋巴细胞增殖和凝集红细胞的活性。3体外抗马立克肿瘤细胞系MSB-1的初步研究以马立克氏病肿瘤细胞系-MSB-1为体外研究对象,分别将ChMIL-18蛋白、HN蛋白、HN-ChMIL18融合蛋白与MSB-1细胞共培养后,通过增殖抑制试验、琼脂糖凝胶电泳、流式细胞仪等方法探讨其抑制机制,MTT结果表明HN蛋白、HN-ChMIL18融合蛋白对MsB-1细胞的增殖有抑制作用,并呈剂量相关性,而ChMIL-18蛋白无抑制作用;琼脂糖凝胶电泳分析发现,ChMIL-18蛋白、HN蛋白、HN-ChMIL18融合蛋白与MSB-1细胞共培养一段时间后,HN蛋白、HN-ChMIL18融合蛋白均出现DNA断裂现象;ChMIL-18蛋白、HN蛋白、HN-ChMIL18融合蛋白与MSB-1细胞共培养后,流式细胞仪检测发现HN蛋白、HN-ChMIL18融合蛋白均引起MSB-1细胞出现了凋亡,而且HN-ChMIL18融合蛋白引起的凋亡数量比HN蛋白多,而单独的ChMIL18不能引起MSB-1细胞凋亡。更多还原

【Abstract】 Newcastle disease virus(NDV),a kind of oncolytic virus,can replicate and proliferate in the tumor cells but not in the normal cells,thus specifically kill tumor cells.With the in-depth study on anti-tumor mechanism of NDV,it has been thought that NDV hemagglutinin- neuraminidase(HN)protein plays an important role in anti-tumor activity induced by NDV.Interleukin-18(IL-18)has the potential to be used as an immunomodulator in the therapy of malignant tumors and cancer gene therapy.To discuss the effect and mechanism against cancer of ChMIL-18 protein,HN protein and HN-ChMIL18 fusion protein,in this study we constructed expression plasmids(pPICZαA-ChMIL 18,pPICZαA-HN,pPICZαA-HN-ChMIL18).Then the expression and the bioactivities of ChMIL-18 protein,HN protein and HN-ChMIL18 protein were detected in vitro.In this study we used MDCC- MSB-1(MD lymphoblastoid cell line)in vitro to explore the inhibitive effect of ChMIL-18 protein, HN protein and HN-ChMIL18 fusion protein on the growth of MSB-1 cell and its mechanism.Both HN protein and HN-ChMIL18 fusion protein could inhibit the growth of MSB-1 and the effect induced by HN-ChMIL18 fusion protein was stronger than that of HN protein.In addition,the possible mechanisms were further discussed,HN protein can induce apoptosis of tumor cells,and ChMIL-18 can enhance the non-specific anti-tumor immunoresponse.ChMIL18 protein can promote the Proliferation of lymphocytes determined by MTT(3-(4,5- dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromid)method,but no inhibitory action MSB-1.It was built foundation for the effect and mechanism against cancer of HN-ChMIL18 fusion protein in vivo,so it has theoretical as well as practical significance.1.Obtain target DNA and Bio-informatics Analysis 1.1 Obtaining ChMIL18 Gene and Bio-informatics AnalysisA pair of primers specific to chicken mature IL-18 were designed and synthesized according to GenBank,to amplify chicken mature IL-18 cDNA by reverse transcription polymerase chain reaction(RT-PCR)from peripheral blood monocyte of chicken.It was cloned into pMD18-T vector and identified by sequencing.The results showed that this gene contains 507bp encoding 169 amino acids with a predicted molecular weight of 19.5KD and pI of 6.36.Bioinformatics analysis indicated that the sequence contained antigenicity but no glycosylation sites.All the results were consistent with those of ChIL18 gene abord.It is necessary to reconstruct ChMIL18 for a high level expression of ChIL18 in Pichia pastoris.By using long-distance inverse PCR,the codon of Arg(CGA)of 28 site and 37 site of ChIL18 were mutated synonymously to Arg(AGA),which is a bias code of Yeast.The mutated pMD18-T-ChMIL18 was transformed into JM109 strain.The mutated ChIL18 gene clone was obtained successfully by digesting,PCR,sequencing and blasting.1.2 Obtaining HN Gene and Bio-informatics AnalysisA cDNA fragment of hemagglutinin-neuraminidae(HN)was obtained from Newcastle disease virus(NDV)LaSota strain by RT-PCR.A 1.7 Kb fragment was amplified,which conformed to the NDV-HN gene fragment’s reported in the Genbank, and cloned into the pMD18-T vector.The recombinant plasmid was proved to be true by enzyme,PCR and sequencing.The sequence and biochemical character are analyzed with Protean.This polypeptide is consist of 577 aa,63.0KD m.w..Its isoelectric point is 7.54. The structure of protein is analyzed which contained 12 Cysteine Residues,5 the potential glycosylation site.Compared HN gene sequence with the corresponding sequence of other strains B1,Clone30,LaSota and so on,the homology of the nucleotide sequencewas 98.8%-99.8%,the homology of the protein sequencewas 98.8%-99.5%1.3 Obtaining HN-ChMIL18 Fusion GeneAccording to MCS of pPICZαA and pBluescript SK+ vector,two pairs of primers with an adapter with were designed.Fusion protein gene of HN and ChMIL18 was separately synthesized and cloned with pMD18-HN、pMD18-ChIL18 as template.DNA fragment was respectively inserted into pMD18-T Vector,and transformed into E.coli JM109,Recombinants were grew in LB culture medium plate and screened.Plasmid DNA was amplified by using PCR and identified by sequening.The results showed that pMD 18-T-HN~f and pMD 18-T-ChMIL 18~f were constructed successfully.pBS SK+ and pMD 18-T-HN~f were digested separately by KpnI and BamHI enzyme, and were linked under T4 DNA Ligase,pBS SK+-HN was constructed and transformed to E.coli JM109,the recombinant was digested by KpnI and BamHI and PCR,the results showed that recombinant plasmid pBS SK+-HN was constructed.And then,pBS SK+-HN and pMD18-T-ChMIL18~f were digested separately by BamHI and NotI enzyme, and were linked under T4 DNA Ligase,pBS SK+-HN-ChMIL18 was constructed and transformed to E.coli JM109,the recombinant was digested by KpnI and NotI and PCR, the results showed that HN-ChMIL18 fusion gene fragment was inserted into pBS SK+.2 Construction of Expression Vector,Inducible Expression and Bioactivities Test of the Recombinant Protein2.1 Construction of pPICZαA-ChMIL18 Vector,Expression and Bioactivities Test of ChMIL18 ProteinSecreted expression vector pPICZαA and pMD18-T-ChMIL18 were digested separately by EcoRI and KpnI enzyme,and were linked under T4 DNA Ligase, pPICZαA-ChMIL18 was constructed and transformed to E.coli JM109,the recombinant was digested by EcoRI and KpnI and PCR,the results showed that recombinant plasmid pPICZαA-ChMIL18 was constructed.ChMIL18 gene fragment was transformed into P.Pastoris X-33 strain by electroporation after pPICZαA-ChMIL18 was lined by SacI enzyme.High-copied transformants were obtained by Zeocin screening.P.pastoris X-33/ pPICZαA-ChMIL18 was expressed under the induction of 1%methanol.SDS-PAGE showed that after being induced with 1%methanol for 4d,the expressed products existed in supernatant and it’s molecular weigh was about 23KD,Western-blot showed good antigenicity and specificity of expressed product,lymphocyte transformation test showed ChMIL18 significantly influenced increment reactivity of lymphocyte of chicken. 2.2 Construction of pPICZαA-HN Vector,Expression and Bioactivities Test of HN ProteinSecreted expression vector pPICZαA and pMD 18-T-HN were digested separately by KpnⅠand NotI enzyme,and were linked under T4 DNA Ligase,pPICZαA-HN was constructed and transformed to E.coli JM109,the recombinant was digested by KpnI and NotI and PCR,the results showed that recombinant plasmid pPICZαA-HN was constructed.HN gene fragment was transformed into P.Pastoris X-33 strain by electroporation after pPICZαA-HN was lined by SacI enzyme.High-copied transformants were obtained by Zeocin screening.P.pastoris X-33/pPICZαA-HN was expressed under the induction of 1%methanol.SDS-PAGE and Western-blot showed that after being induced with 1%methanol,the expressed products existed in supematant and it’s molecular weigh was about 97KD,has good antigenicity and specificity of expressed product,hemagglutination test showed HN protein could absorb chicken red blood cells(RBC).2.3 Construction of pPICZαA-HN-ChMIL18 Vector,Expression and Bioactivities Test of HN-ChMIL18 Fusion ProteinSecreted expression vector pPICZαA and pBS SK+-HN-ChMIL18 were digested separately by KpnI and NofI enzyme,and were linked under T4 DNA Ligase, pPICZαA-HN-ChMIL18 was constructed and transformed into E.coli JM109,the recombinant was digested by KpnⅠand NotI and PCR,the results showed that recombinant plasmid pPICZαA-HN-ChMIL18 was constructed.HN-ChMIL18 gene fragment was transformed into P.Pastoris X-33 strain by electroporation after pPICZαA- HN-ChMIL18 was lined by SacI enzyme.High-copied transformants were obtained by Zeocin screening.P.pastoris X-33/pPICZαA-HN-ChMIL18 was expressed under the induction of 1%methanol.SDS-PAGE and Western-blot showed that the expressed products existed in supematant and it’s molecular weigh was about 103KD,has good antigenicity,lymphocyte transformation test and Hemagglutination test showed HN-ChMIL18 protein significantly influenced increment reactivity of lymphocyte of chicken and absorb chicken red blood cells(RBC).3 Study on anti-MDCC-MSB-1 in vitroIn this study we used MDCC- MSB-1(MD lymphoblastoid cell line)in vitro to explore the inhibitive effect of ChMIL-18 protein,HN protein and HN-ChMIL18 fusion protein on the growth of MSB-1 cell and its mechanism.The inhibitive effect of ChMIL-18 protein,HN protein and HN-ChMIL18 fusion protein on MSB-1 cell by MTT assay,and explored the mechanism through agarose gel electrophoresis and flow cytometry.MTT assay showed that HN protein and HN-ChMIL18 fusion protein inhibited the growth of MSB-I cell,Agarose gel electrophoresis showed that MSB-1 cell treated with HN protein and HN-ChMIL18 fusion protein for 72 hours had DNA ladder.Flow cytometry analysis indicated that apoptotic cells appeared after MSB-1 cell treated with HN protein and HN-ChMIL18 fusion protein for some time.These results suggested that HN protein and HN-ChMIL18 fusion protein inhibited the growth of MSB-1 cell,and the mechanism was relative to inhibiting the synthesis of DNA and inducing apoptosis of some MSB-1,However,ChMIL-18 didn’t so.更多还原