鸡白细胞介素18基因的克隆与原核表达
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摘要
白介素18(IL-18)最早被称为干扰素诱导因子(interferon-γ-inducing factor,IGIF),是近年发现的一种重要的免疫调节因子。该实验根据Gen Bank中已公布的鸡IL-18的基因序列,设计并合成了一对特异性引物,应用RT-PCR方法扩增出IL-18基因,并将该基因克隆到p MD18-T载体中,经过质粒酶切、PCR和测序鉴定,阳性克隆命名为Chp MD18-T-IL-18。将Chp MD18-T-IL-18双酶切后与p ET32a表达载体连接,转化到大肠杆菌BL21感受态细胞中,经IPTG诱导表达鸡IL-18蛋白,结果表明,在28k D左右出现了目的条带,经west-ern blot检测确定该蛋白获得高效表达,为进一步研究该蛋白的功能奠定了基础。
Interleukin 18(IL-18)is called interferon gamma inducing factor(IGIF).It is a kind of important immuneadjustment factor.In this paper,a pair of specific primer was designed and synthesised according to IL-18 DNA se-quence in Gen Bank,IL-18 gene was amplified by RT-PCR method and was inserted into p MD18-T vector.Positiveplasmids were identified by PCR and restriction enzyme digestion methods.Then IL- 18 gene was inserted intop ET32 a vector.Positive plasmids were identified by PCR and restriction enzyme digestion methods and were trans-formed into escherichia coli competent cells.Recombinant E.coli were induced by IPTG and IL-18 protein was de-tected by SDS-PAGE and Western blot methods.The results showed IL-18 protein was expressed.This would estab-lish a basis for studying the function of this protein.
Interleukin 18(IL-18)is called interferon gamma inducing factor(IGIF).It is a kind of important immuneadjustment factor.In this paper,a pair of specific primer was designed and synthesised according to IL-18 DNA se-quence in Gen Bank,IL-18 gene was amplified by RT-PCR method and was inserted into p MD18-T vector.Positiveplasmids were identified by PCR and restriction enzyme digestion methods.Then IL- 18 gene was inserted intop ET32 a vector.Positive plasmids were identified by PCR and restriction enzyme digestion methods and were trans-formed into escherichia coli competent cells.Recombinant E.coli were induced by IPTG and IL-18 protein was de-tected by SDS-PAGE and Western blot methods.The results showed IL-18 protein was expressed.This would estab-lish a basis for studying the function of this protein.
引文
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[9]何静,张盼盼,孙敏华,等.鸡ɑ干扰素/白细胞介素18基因融合表达和抗病毒活性研究[J].华南农业大学学报,2015,36(1):18-22.
[10]赵文博,李瑞航,贺鹏亮,等.羊口疮病毒B2L基因克隆及表达[J].黑龙江八一农垦大学学报,2015,27(2):42-45.
[11]Kim S H,Cho D,Hwang S Y,et al.Eff icie nt induction of antigen specific T helper type 1-mediated immune re sponses by intramuscular injection with ovalbumin/interleukin-18 fusion DNA[J].Vaccine,2001,19:4107-4114.
[12]Marshall DJ,Rudnick KA,Mc Carthy SG,et al.Interleukin-18enhances Th1 immunity and tumor protection of a DNA vaccine[J].Vaccine,2006,24:244-253.
[13]Billaut-Mulot O,Idziorek T,Loyens M,et al.Modulation of cellular and humoral immune responses to amultiepitopic HIV-1DNA vaccine by interleukin-18 DNA immunization/viral protein boost[J].Vaccine,2001,19:2803-2811.
[2]Srivastava S,Salim N,Robertson MJ.Interleukin-18:biology and role in the immunotherapy of cancer.Curr Med Chem,2010,17:3353–3357.
[3]Tschoeke SK,Oberholzer A,Moldawer LL.Interleukin-18:a novel prognostic cytokine in bacteria-induced sepsis[J].Crit Care Med,2006,34:1225–1233.
[4]Lee HR,Yoon SY,Song SB,et al.Interleukin-18-mediated interferon-gamma secretion is regulated by thymosin beta 4 in human NK cells[J].Immunobiology,2011,216:1155-1162.
[5]Tse BW,Russell PJ,Lochner M,et al.IL-18 inhibits growth of murine orthotopic prostate carcinomas via both adaptive and innate immune mechanisms[J].Plo S One,2011,6:24241.
[6]Tian H,Shi G,Yang G,et al.Cellular immunotherapy using irradiated lung cancer cell vaccine co-expressing GM-CSF and IL-18 can induce significant antitumor effects[J].BMC Cancer,2014,14:48.
[7]Wong JL,Muthuswamy R,Bartlett DL,et al.IL-18-based combinatorial adjuvants promote the intranodal production of CCL19by NK cells and dendritic cells of cancer patients[J].Oncoimmunology,2013,2:26245.
[8]唐梦君,王红宁,周生,等.IBV M基因与IL-18基因共表达DNA疫苗的免疫原性[J].中国兽医学报,2008,28(7):757-761.
[9]何静,张盼盼,孙敏华,等.鸡ɑ干扰素/白细胞介素18基因融合表达和抗病毒活性研究[J].华南农业大学学报,2015,36(1):18-22.
[10]赵文博,李瑞航,贺鹏亮,等.羊口疮病毒B2L基因克隆及表达[J].黑龙江八一农垦大学学报,2015,27(2):42-45.
[11]Kim S H,Cho D,Hwang S Y,et al.Eff icie nt induction of antigen specific T helper type 1-mediated immune re sponses by intramuscular injection with ovalbumin/interleukin-18 fusion DNA[J].Vaccine,2001,19:4107-4114.
[12]Marshall DJ,Rudnick KA,Mc Carthy SG,et al.Interleukin-18enhances Th1 immunity and tumor protection of a DNA vaccine[J].Vaccine,2006,24:244-253.
[13]Billaut-Mulot O,Idziorek T,Loyens M,et al.Modulation of cellular and humoral immune responses to amultiepitopic HIV-1DNA vaccine by interleukin-18 DNA immunization/viral protein boost[J].Vaccine,2001,19:2803-2811.