鸡白细胞介素18成熟蛋白在昆虫杆状病毒系统中的表达及其活性检测

Expression of Chicken Interleukin-18 Maturation Protein Gene in Insect Cells and Identification of Bioactivity of Its Expressed Protein

作者: 专业:预防兽医学 导师:胡敬东 年度:2010 学位:硕士  院校: 山东农业大学

Keywords

chicken interleukin-18 mature protein, baculovirus expression vector system, insect cells, silkworm baculovirus expression vector system, bioactivity

        白细胞介素18(Interleukine-18, IL-18)是1995年由Okmura等从小鼠肝脏中克隆获得的一种细胞因子,主要由单核巨噬细胞系统的细胞分泌,具有强烈的IFN-γ诱生能力,对机体起着重要的免疫调节和保护作用,在抗微生物感染、抗肿瘤免疫中具有应用潜力。对人和哺乳动物IL-18的研究已有许多报道,而鸡IL-18(ChIL-18)基因发现得比较晚,由于它具有与人和哺乳动物相似的生物学功能,因此ChIL-18在比较免疫学研究和禽病治疗中具有重要意义。本实验室从2001年开始进行鸡白细胞介素18的研究,已经在原核系统中实现了鸡白细胞介素18成熟蛋白(mChIL-18)基因的高效表达,但由于该系统表达的蛋白存在非糖基化形式等缺陷,使得表达的蛋白分子常失去天然结构,因而对表达的重组蛋白的活性还存在争议。本试验利用昆虫/杆状病毒系统表达鸡白细胞介素18成熟蛋白,进而对该重组蛋白有效地进行了生物学活性检测。参考已发表的mChIL-18的cDNA基因序列,设计一对特异性引物,以pMD18-T-mChIL18质粒为模板,扩增出编码mChIL-18成熟蛋白基因的cDNA片段。再以杆状病毒pFastBac TM HTb为载体,将mChIL-18基因插入到表达载体pFastBacTM HTb的多角体蛋白启动子PPH的下游,构建重组转移载体质粒pFastBacTM HTb-mChIL-18,然后将已构建好的重组转移载体质粒转座入大肠杆菌DH10BacTM感受态细胞中,经过三次蓝白斑筛选纯化后提取质粒,通过一对通用引物M13(Bacmid含有该引物Forward和Reverse两个引物位点,能够从两侧扩增LacZα互补区域内的mini-attTn7位点,有利于PCR分析)进行PCR扩增鉴定,得到重组Bacmid(Bacmid-mChIL18)。在转染试剂Cellfectin的作用下,通过脂质体介导法将纯化的Bacmid-mChIL-18转染至草地贪夜蛾细胞(Spodoptera frugiperda 9,sf9)获得P1代重组杆状病毒,用P1代病毒反复感染sf9来扩增病毒滴度,将达到一定滴度(107~108pfu/mL)的重组杆状病毒再感染sf9,并在感染后不同时间段收获sf9细胞及培养上清,然后对细胞进行超声波破碎,离心后分别取裂解上清和沉淀,经SDS-PAGE电泳分析、Western blotting和间接免疫荧光(IFA)检测,结果表明,mChIL-18基因在昆虫细胞sf9中获得了表达,表达的目的条带分子量约为23KD。采用鸡脾淋巴细胞增殖试验(MTT法)、IFN-γ诱导实验和水疱性口炎病毒(VSV)抑制试验对表达的蛋白进行生物学活性测定。鸡脾淋巴细胞增殖试验表明,mChIL-18融合蛋白能够明显促进淋巴细胞的增殖,不同浓度的蛋白均可刺激淋巴细胞转化。当浓度为200ng/mL时,刺激转化效果最佳,但随着蛋白浓度的增大,淋巴细胞的增殖指数则会降低;VSV病毒活性抑制试验表明,当蛋白浓度大于100ng/mL时能刺激脾淋巴细胞产生IFN-γ,并且随着mChIL-18蛋白浓度的增加,诱导产生IFN-γ的量随之增加;将不同稀释度的IFN-γ分别作用于VSV,在IFN-γ≥1×102U/mL时,具有较强的抑制效果,当IFN-γ为10 U/mL时,只能达到50%的保护。该结果说明mChIL-18融合蛋白的抗病毒活性是通过IFN-γ实现的,且在一定浓度范围内具有抑制VSV病毒产生细胞病变的作用。
    Interleukin-18(IL-18), a novel discovered cytokine, which is the potent inducor of IFN-γproduction, was firstly cloned by Okamura from Hepar in 1995 and mainly secreted from cells of monocyte macrophage system. It has been demonstrated that IL-18 plays a major role in immunoregulation and protection, and has large potential in host defense against various micro- organic infection and tumor.These research on human beings and animals were identified by many documents recently. However, the study on chicken interleukin-18(ChIL-18) lagged off the mammal interleukin-18. Previously, the gene of ChIL-18 was cloned, but further study is not performed.The discovery of ChIL-18 gene is very meaningful for both the study on comparative immuneology and the therapeutics for poultry diseases.In 2001, the high expression of the mature chicken interleukin- 18(mChIL-18) in E.coli had been finished in our laboratory. But the protein lost natural structure because of the fault of the system, such as noglycosy- lation form.So people have some questions about the bioactivity of the protein expressed by E.coli. In the present study, how to obtain the efficiently expressed mChIL-18 protein with biological activity was studied.In the experiment, the insect/baculovirus system was selected to express the chIL-18 mature protein and the biologic activity was detected. The mature chicken interleukin-18(mChIL-18) gene was amplified by designing one pair of primers according to the sequence which have been published.Recombinant baculovirus transfer vector was constructed by inserting cDNA copy of mChIL-18 gene into the downstreams of PH promoter of the baculovirus expression plasmid pFastBacTM HTb. Finally, the constructing eukaryotic expression plasmid named as pFastBacTM HTb-mChIL-18. After constructing pFastBacTMHTb-mChIL-18 plasmid, we make it transformate into DH10BacTM competent cells. And a recombinant bacmid(Bacmid-ChIL18) was obtained by there times blue/white spot selection and PCR identification by M13 universal primers(The bacmid contains M13 Forward and M13 Reverse priming sites flanking the mini-attTn7 site within the lacZα- complementation region to facilitate PCR analysis). In order to harvest the P1 recombinant aculovirus, we transfected the purified bacmid into Spodoptera frugiperda 9(sf9) by using a cationic lipid such as Cellfectin Reagent. Raised the virus titre by infecting sf9 many times and infected the sf9 with the higher baculoviral stock(107~108 pfu/mL) for expressing protein and harvested the supernatant and cells in different times. The expressed mChIL-18 protein was analyzed by SDS– PAGE, detected by Western blotting and IFA, and the results demonstrated that recombinant protein of 23kDa in molecular mass was expressed successfully in insect cells.The biological activities of purified fusion protein, including of the proliferation of lymphocyte, inducing IFN-γproduction and inhibiting the Vesicular stomatitis virus(VSV) activity, were detected.The experiment of MTT showed that every concentration of mchIL-18 fusion protein could enhance proliferation of lymphocyte, and the effect was best when it was 200ng/mL and the proliferation exponent was 2.87. The experiment of VSV inhibition indicated that the mchIL-18 fusion protein could induce spleenocytes to produce IFN-γwith high activity when it was more than 100ng/mL, and the amount of mChIL-18 fusion protein and its inducing effect on IFN-γhad a direct proportion. In the experiment of VSV inhibition, different dilution of IFN-γwas used and it could protect the cells when it was more than 10U/mL. The result showed that mChIL-18 fusion protein could inhibit the VSV activity by inducing IFN-γproduction in spleen lymphocyte. So it can be concluded that mChIL-18 was expressed in sf9 and the expression product has functional activities.
        

鸡白细胞介素18成熟蛋白在昆虫杆状病毒系统中的表达及其活性检测

中文摘要12-14
英文摘要14-15
1 引言16-39
    1.1 白细胞介素-18的研究进展16-27
        1.1.1 IL-18 的发现16
        1.1.2 IL-18 的分布及产生16-17
        1.1.3 IL-18 的基因结构和基因表达调控17-18
        1.1.4 IL-18 的受体与信号转导18-19
        1.1.5 IL-18 的生物学功能19-27
    1.2 鸡白细胞介素18 的研究进展27-29
        1.2.1 鸡白细胞介素18 的发现27
        1.2.2 鸡白细胞介素18 的研究现状27-29
    1.3 昆虫杆状病毒表达载体系统研究进展29-38
        1.3.1 昆虫杆状病毒的分子生物学30-32
        1.3.2 昆虫杆状病毒表达系统研究的新进展32-34
        1.3.3 昆虫杆状病毒表达载体系统的优越性34-35
        1.3.4 昆虫杆状病毒表达载体系统的应用35-38
    1.4 研究的目的与意义38-39
2 材料与方法39-60
    2.1 材料39-45
        2.1.1 菌株与质粒39-40
        2.1.2 细胞、受体菌及细胞培养基40-41
        2.1.3 主要试剂41
        2.1.4 培养基及主要试剂的配制方法41-44
        2.1.5 仪器与设备44-45
    2.2 方法45-60
        2.2.1 引物设计及合成46
        2.2.2 mChIL-18 成熟蛋白基因的PCR 扩增46-47
        2.2.3 mChIL-18 重组转移载体的构建及鉴定47-51
            2.2.3.1 PCR 产物的回收与纯化47-48
            2.2.3.2 PCR产物与转移载体pFastBacTM HTb连接48
            2.2.3.3 DH5α感受态细胞的制备方法48
            2.2.3.4 连接产物转化DH5α感受态细胞48-49
            2.2.3.5 重组质粒的鉴定49-51
        2.2.4 重组质粒的转座(即构建重组Bacmid)51-53
            2.2.4.1 DH10Bac~(TM) 感受态细胞的制备51
            2.2.4.2 重组质粒转座DH10Bac 感受态细胞51-52
            2.2.4.3 重组BacmidDNA的提取及鉴定52-53
        2.2.5 重组Bacmid 转染sf9昆虫细胞及收获重组杆状病毒53-57
            2.2.5.1 重组Bacmid转染sf9昆虫细胞53-54
            2.2.5.2 重组杆状病毒的收获及其DNA的提取与鉴定54-55
            2.2.5.3 扩增P1代病毒原液55-56
            2.2.5.4 噬斑测定病毒滴度56-57
        2.2.6 mChIL-18在sf9昆虫细胞中的表达及其检测57-58
            2.2.6.1 m ChIL-18在sf9昆虫细胞中的表达57
            2.2.6.2 SDS-PAGE及Western-blotting检测57
            2.2.6.3 IFA检测57-58
        2.2.7 表达产物的生物学活性测定58-60
            2.2.7.1 mChIL-18融合蛋白促进鸡脾淋巴细胞增殖试验(MTT法)58-59
            2.2.7.2 mChIL-18融合蛋白诱导鸡脾淋巴细胞产生IFN-γ59
            2.2.7.3 mChIL-18融合蛋白诱导的IFN-γ对VSV的抑制活性59-60
3 结果与分析60-73
    3.1 mChIL-18成熟蛋白基因的PCR扩增60-61
    3.2 重组转移载体的构建及鉴定61-63
        3.2.1 重组转移载体的PCR和双酶切鉴定61-63
        3.2.2 重组转移载体的序列测定63
    3.3 重组Bacmid 的鉴定63-65
    3.4 重组杆状病毒的 DNA 鉴定65
    3.5 mChIL-18 在sf9 昆虫细胞中的表达及检测65-71
        3.5.1 表达产物的SDS-PAGE 分析65-69
        3.5.2 表达产物的Western blotting检测69-70
        3.5.3 表达产物的IFA检测70-71
    3.6 mChIL-18蛋白生物学活性的测定71-73
        3.6.1 mChIL-18融合蛋白促进鸡脾淋巴细胞增殖试验71-72
        3.6.2 mChIL-18融合蛋白诱导鸡脾淋巴细胞产生IFN-γ72
        3.6.3 mChIL-18融合蛋白诱导的IFN-γ对VSV的抑制作用72-73
4 讨论73-78
    4.1 mChIL-18 重组转移载体的构建及鉴定73-75
    4.2 mChIL-18 在sf9 昆虫细胞中的表达及其检测75-76
    4.3 表达产物的生物学活性测定76-78
5 结论78-79
参考文献79-90
附录90-93
致谢93-94
攻读学位期间发表论文情况94
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