鸭α干扰素原（真）核表达及其生物学活性研究

【作者】 陈斌；

【导师】 程安春；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2008， 博士

【摘要】 本文对四川省内地方品种四川麻鸭的干扰素进行如下系列研究:对四川麻鸭Ⅰ型干扰素基因IFN-αORF进行克隆、鉴定和序列分析;构建IFN-αORF基因原核表达载体和真核表达载体;IFN-α原核表达及其生物学活性研究;利用雏鸭为模型,应用免疫组织化学方法研究pcDNA-SDIFN-α真核表达质粒在鸭体内的动态表达分布规律;同时对作为免疫佐剂的pcDNA-SDIFN-α真核表达质粒免疫调节活性进行研究,获得以下结果:1.四川麻鸭IFN-αORF基因克隆、序列比对分析和蛋白结构预测应用Oligo6.0软件设计了两对加入EcoRI和BamHI两个酶切位点的特异性引物对,采用PCR和nest-PCR的方法,从鸭肝脏组织基因组DNA扩增得到大小两个完全包含麻鸭α干扰素ORF基因的DNA片段。其中小片段更适合用于表达,并将其进行pGEM-T载体克隆与测序,对测序结果采用ClustalX软件进行序列分析以及与北京鸭、鸡、鹅等NCBI上序列进行了比较。结果显示四川麻鸭与北京鸭ORF基因序列十分相似,其核苷酸同源性达到99.13%,氨基酸同源性为98.96%。与鸡的核苷酸及氨基酸同源性分别为71.8%、49.97-50.47%,与鹅的核苷酸同源性为96%。同时用BioEdit、NetNGlyc、TreeView软件对该基因所推测的蛋白进行了分析,结果表明四川麻鸭有2个潜在糖基化位点,1个蛋白激酶C磷酸化位点(Protein kinase C phosphorylation)和1个酪蛋白激酶Ⅲ磷酸化位点(Casein kinaseⅢphosphorylation site),信号肽切割位点28-29位ANA和FS氨基酸之间,成熟的鸭α干扰素有5个疏水区,无跨膜区。2.SDIFN-αORF基因原核表达载体pBV220-SDIFN-α的构建及表达产物的抗病毒活性采用BamHI和EcoRI酶切,通过定向粘末段连接法,构建了原核表达载体pBV220-SDIFN-α质粒。并将质粒转化到原核表达菌株DH5α中进行表达,采用温度诱导的方式,对诱导后的菌液进行SDS-PAGE检测。结果显示工程菌进行了表达,产生的包涵体蛋白分子量为40KD左右。对纯化后的包涵体采用稀释复性的方法复性,采用VSV/DEF系统初步检测抗病毒活性,干扰素的抗病毒活性约为150U/ml,比活力为440U/mg。应用FQ-PCR检测方法,动态监测体内抑制DHBV和体外抑制DPV病毒核酸。结果发现,表达的干扰素具有强烈的病毒复制抑制作用,具有明显的生物学活性。3.SDIFN-αORF基因真核表达载体pcDNA-SDIFN-α的构建及鉴定利用合成在引物两端的EcoRI、BamHI酶切位点,将目的基因成功地从pGEM-T载体克隆重组体中切下,并通过粘端连接的方法,连接到pUC18定向位置上,然后用EcoRI和XbaI双酶切真核表达载体pcDNA3.1(+)和pUC18-SDIFN-α,实现了SDIFN-α定向克隆到pcDNA3.1(+)载体上。四川麻鸭IFN-α真核表达重组质粒pcDNA-SDIFN-α经BamHI、EcoRI和XbaI三种酶切验证,并测序证明四川麻鸭IFN-α的真核表达质粒pcDNA-SDIFN-α构建正确。4.免疫组织化学检测真核表达质粒pcDNA-SDIFN-α在鸭体内表达方法的建立及动态表达规律的研究以pcDNA-SDIFN-α真核质粒免疫鸭,分时间段采集鸭16种不同组织,建立了免疫组织化学检测方法,并应用该方法检测干扰素在体内的动态表达规律。pcDNA-SDIFN-α免疫鸭后,持续表达时间长达9周。能在肺脏细胞、心肌细胞、肝细胞、脾细胞、肠腺和肠绒毛细胞、肌肉细胞、皮肤内检测到表达产物,在14d-35d进行了高峰表达。胸腺、胰腺、法氏囊和哈德氏腺未检测到阳性黄色颗粒,肺脏、免疫部位最早出现阳性颗粒,脑组织细胞中阳性颗粒数量随时间变化较小。其中基因枪免疫鸭后,各组织中的含量和表达量呈现的总体规律为6μg组＞3μg组＞1μg组。肌肉注射免疫鸭后,各组织中的含量和表达量呈现的总体规律为200μg组＞100μg组＞50μg组,都具有一定的剂量相关性。基因枪轰击法较肌肉注射法IFN-α基因表达出现的时间早,3h就可以检测到表达产物。同时,相对于肌肉注射法免疫,基因枪轰击法所用的表达质粒用量更少。5.真核表达质粒pcDNA-SDIFN-α在鸭体内免疫调节剂活性的初步研究以pcDNA-SDIFN-α真核表达质粒为免疫调节剂,研究对DPV/DVH弱毒苗免疫活性调节作用。分不同时间段采血,采用MTT法测定鸭外周血T淋巴细胞转化效果、CD3+T淋巴细胞数量变化和特异性DPV中和抗体IgG水平。结果表明,一定剂量的pcDNA-SDIFN-α真核表达质粒作为免疫调节剂在一定时间内能促进和提高细胞免疫和体液免疫应答,发挥着有效的正向免疫调节作用。更多还原

【Abstract】 In this paper,the study is on Sichuan Province local varieties of duck interferon. We conducted the following series of studies:Sichuan duck typeⅠinterferon IFN-αORF gene sequence was cloned、identified and analysis;IFN-αORF prokaryotic gene expression vector and eukaryotic expression vector were successful constructed; interferon-αhas been expressed by prokaryotic expression &.biological activity;Useing ducklings as model,by means of immunohistochemistry.We studied on pcDNA-SDIFN-αvector dynamic distribution of expression in ducks;At the same time as the immunoadjuvant,we test the pcDNA-SDIFN-αeukaryotic expression vector to carry on immunological regulation function in ducks.The results were as follows:1.Sichuan duck IFN-αORF gene cloning,sequencing,analysis and protein structure prediction.Application software Oligo6.0,designed two pairs of specific primers within BamHI and EcoRI restriction sites,by PCR and nest-PCR method,we get two size amplified from duck liver geome about completely duck interferon-αgene ORF DNA fragment,Small fragments of which is more suitable for expression.Using pGEM-T vector,we get clone about duck interferon-αgene.Using ClustalX sequence analysis software,as well as with beijing ducks,chickens,geese,and other sequences in the NCBI comparison,The results showed that Sichuan and Beijing duck ORF sequences are very similar,its nucleotide homology to 99.13%,amino acid homology of 98.96%.And chicken nucleotide and amino acid homology are 71.8%and 49.97-50.47%,the goose nucleotide homology of is 96%.At the same time using BioEdit,NetNGlyc,TreeView software speculate that the gene protein was analyzed.The results show that Sichuan duck has two potential glycosylation sites,a protein kinase C phosphorylation sites,and a casein kinaseⅡphosphorylation sites,the signal peptide cleavage 28-29(ANA and FS) between amino acids,and mature duck interferon-αhas five hydrophobic,no transmembrane region.2.SDIFN-αORF prokaryotic gene expression vector pBV220-α-SDIFN Construction, expression and the anti-viral activity detection.BamHI and EcoRt restriction,by the end of visco-directional link,prokaryotic expression vector pBV220-SDIFN-αplasmid was constructed.And the plasmid was transformed into prokaryotic expression strain Ecoli DH5α,using temperature-induced and SDS-PAGE.The results showed that product is 40 KD.By purified and refolding and VSV /DEF system,we detected the anti-viral activity of product.The results of antiviral activity are about 150 U / ml,vitality about 440 U / mg.Application FQ-PCR method,dynamic monitoring of the virus nucleic acid about DHBV in vivo and DPV in vitro.The results showed that the interferon has a strong inhibition of virus amplify.3.SDIFN-αORF eukaryotic expression vector pcDNA-SDIFN-αconstruction and identification.By means of primers with ends of the EcoRI &.BamHI restriction sites,we get gene from the pGEM-T vector,and link with the pUC 18 directional positions.And then use EcoRI &.Xbal double digestion of eukaryotic expression-vector pcDNA3.1(+) and pUC 18-SDIFN-α,we get pcDNA-SDIFN-αeukaryotic expression vector clone.By BamHI, EcoRI and XbaI three digestions and sequencing shows that it is correct about Sichuan duck IFN-αeukaryotic expression recombinant pcDNA-SDIFN-αplasmid.4.Immunohistochemistry’s Established and reasearch on eukaryotic expression plasmid pcDNA-SDIFN-αexpression laws in duck.Using pcDNA-SDIFN-αeukaryotic vector immune duck,the 16 different duck organizations were collected at the different time.We established the immunohistochemistry method.And application the method,we tested the expression laws of interferon in vivo.pcDNA-SDIFN-αimmune duck,continued to express time up to nine weeks.In the lung cells,heart muscle cells,liver cells and spleen cells,intestinal glands and intestinal villi cells,muscle cells,skin cells successfully expressed,14 d-35d in a peak of expression.Thymus,pancreas,Bursa and Bemhard’s glands were not detected yellow particles.The earliest site is in Lung &.immune tissue.Brain tissue’s Positive reaction is almost no change with times.By Gene gun immunization method,the content of exprcssion shpws of Group 6μg＞3μg group＞1μg group.Intramuscular immunization ducks,the content of expression shows of 200μg group＞100μg group＞50μg group, the all of which have a dose related.Gene gun immunization method is earlier than intramuscular gene in expression;3 h can be detected expression products by Gene gun immunization method.At the same time,comparing to intramuscular injection method,the gene gun use less plasmids.5.The study on Eukaryotic expression plasmid pcDNA-SDIFN-αas immune adjuvant.pcDNA- SDIFN-αeukaryotic expression vector as for immune adjuvant,research on the DPV / DVH attenuated vaccine regulation of immune activity.At different times,by MTT assay determination of T lymphocytes,the number of CD3 + T lymphocytes and specific changes in the IgG antibody levels about DPV immuned duck.The results show that a certain dose of pcDNA-SDIFN-αeukaryotic expression vector as an immune adjuvants have positive role in regulating.更多还原