摘要
白细胞介素18是一种新发现的细胞因子,具有多种生物学功能,能够促进γ干扰素(IFN-γ)的产生,刺激淋巴细胞转化,增强NK细胞的杀伤活性,在介导细胞免疫、抵抗微生物感染方而具有重要的作用。同时,白细胞介素18在作为天然的免疫调节剂,在集约化畜禽养殖业中,是一种可以替代传统疗法的新型免疫调节剂,具有巨大的应用前景。目前国内外对IL-18的研究主要集中在人和鼠等哺乳动物,而对鸡IL-18的研究报道不多,为此,本试验应用已成功开发使用的原核大肠杆菌表达体系及酵母表达系统对鸡IL-18进行了表达,并对其表达产物的生物学活性分别进行了分析,为进一步研究鸡IL-18的作用机理及其在鸡体内的生物学功能奠定了基础。
第一部分:鸡IL-18在大肠杆菌BL21-ChIL-18中的表达、纯化及体外生物活性检测首先对重组菌BL21-ChIL-18的遗传稳定性进行了分析,筛选出了其最佳诱导表达条件,然后用S
ephadex G-100对目的蛋白进行纯化,并以牛血清白蛋白为标准蛋白质,用Bradford法检测了纯化的目的蛋白的浓度,对其进行了定量。最后分别用Western bloting、MTT和ELISA等方法在体外分析了其生物学活性。结果表明该重组工程菌能稳定地表达鸡IL-18,在诱导温度37℃时,IPTG浓度为0.6 mmol/L时,诱导4 h目的蛋白相对表达量最高。纯化后的体外活性检测试验表明,其表达的鸡白介素18具有刺激淋巴细胞转化和诱导其分泌γ干扰素的活性。
第二部分:鸡IL-18在真核毕赤酵母的表达、纯化和体外生物学活性检测首先应用PCR技术从重组质粒pMD18-T-ChIL-18中扩增出鸡IL-18成熟肽基因,亚克隆于毕赤酵母表达载体pPICZαA上,构建重组质粒pPICZαA-ChIL-18。经酶切、PCR和测序鉴定正确后,电转化入毕赤酵母菌X-33中,筛选多拷贝单克隆进行诱导表达。表达产物用Western bloting鉴定正确后,对其遗传稳定性进行分析,并对其表达条件进行优化,然后再其最佳诱导条件下对重组工程菌进行大量诱导表达并用S
ephadex G-100对目的蛋白进行纯化,并用Bradford法检测纯化目的蛋白的浓度,对其进行定量,最后分别用MTT和ELISA等方法在体外对其生物学活性进行分析。结果,成功构建了鸡IL-18真核酵母表达载体,且该重组工程菌能稳定地表达鸡IL-18。其在培养液pH6.0,甲醇诱导浓度1.5%,诱导温度为26℃的条件下诱导96 h,目的蛋白的表达量最高。纯化后的体外活性检测试验表明,其表达的鸡白介素18具有刺激淋巴细胞转化和诱导淋巴细胞分泌r干扰素的活性。
第三部分:重组鸡IL-18对新城疫疫苗免疫免疫效果的影响0日龄300只鸡随机分为6组,每组50只鸡,即空白对照组,新城疫疫苗组,新城疫疫苗+原核IL-18组,新城疫疫苗+原核空载体对照组,新城疫疫苗+真核IL-18组,新城疫疫苗+真核空载体对照组。所有免疫组均于8日龄分别免疫接种相应疫苗,并分别于免疫后的第3 d,7 d,14 d,21 d,28 d和35 d各组鸡随即抽取4只,对其血清的抗体效价、γ干扰素含量、外周血的T淋巴细胞转化水平及其NO分泌和免疫器官指数进行测定,18天后各组随机取20只鸡用于动物攻毒保护试验,。结果表明重组鸡IL-18蛋白增强了新城疫疫苗的免疫效果,提高了血清中抗体水平和γ干扰素含量、提高了T淋巴细胞的转化水平和巨噬细胞分泌NO的水平,促进了鸡免疫器官的生长发育和成熟,升高了其各免疫器官指数,且部分指标差异达到显著(0.01﹤P﹤0.05)或极显著水平(P<0.01)。
综上所述,本研究成功的在原核细胞大肠杆菌BL21及真核细胞毕赤酵母X-33中分别表达了鸡IL-18,表达量均较高,且纯化后都具有生物学活性,对新城疫疫苗都有免疫增强作用。
Interleukin-18(IL-18) is a novel cytokine,which has multiple biologicala ctivities including induction of IFN-γfromN Kcells and antigen-or mitogen-stimulated Thl cells,upregulation of IL-2R onT cells,enhancement of Fas ligand-mediated cytotoxicity of T-helper cells and augmentation of NKcell cytotoxicit。At the same time, IL-18, as natural mediators of the immune response,offer exciting alternatives to conventional therapeutics in control of diseases in intensive livestock and poultry industries.And it has great application prospect. Now the study of IL-18 is focused on mammals such as human and mouse in home and abroad, and there is a few about chicken IL-18.So this experiment has successfully used the prokaryotic expression system of E. coli and yeast expression system for chicken IL-18 expression,and has analyzed the biological activity of the expression products.This laid a foundation.for further study of mechanism of chicken IL-18 and its biological function in vivo chicken
Part I: expression, purification and detection of biological activity in vitro of chicken IL-18 in E. coli BL21-ChIL-18 In this part ,we analyzed the genetic stability and selected the best conditions for inducible expression of recombinant strain BL21-ChIL-18,firstly.Then purify the target protein by S
ephadex G-100 purification and. determine the concentration of interest protein by Bradford method.analyzed Last, analyzed its biological activity.of ChIL-18 in vitro by Western blot, MTT and ELISA.The results show that the recombinant bacteria can express chicken IL-18 stably. Under 37℃, induced the recombinant strain BL21-ChIL-18 4 h by 0.6mmol / L ,it can express the highest interest protein. After purified , the expressed chicken IL-18. could induce T lymphocytes to maturation and expressing IFN-γgene in vitro .
Part II: Chicken IL-18 mature p
eptide gene was amplified from the recombinant plasmid pMD18-T-ChIL-18 by PCR ,and was subcloned into Pichia pastoris expression vector pPICZαA to construct the recombinant plasmid pPICZαA-ChIL-18. After identified by restriction enzymes digestion analysis、PCR and DNA sequencing, the recombinant plasmid was transformed into Pichia pastoris X-33 firstly.Then choosing the multi-copy recombinant strains to be induced for expression.Then analyzed the genetic stability and selected the best conditions for inducible expression of recombinant strain X-33-ChIL-18 after it has been ascertained by Western blot.Last, the bioactivity of rchIL-18 was analysed by ELISA and MTT after purified by S
ephadex G-100 column. Results: The chicken IL-18 with the immunogenicity was secreted by Pichia pastoris stably.It could induce T lymphocytes to maturation and expression IFN-γgene in vitro.
Part III: Eeffct of reeombinnat chichen IL-18 on NDV vaccine as immunopotentiator
In the experiment,300 chichens were randomly divided into six groups(50 chichens each group):control group , NDV vaccine gorup , NDV vaecine+ IL-18 of prokaryotic expession(50ug/chichen)group,NDV vaeeine+prokaryotic black expession vector control group , NDV vaccine+IL-18 of Pichia pastoris expression(250ug)group and NDV vaceine+ Pichia pastoris black expession vector control .All groups were immunized by its vaceine respectively on the eighth day Then respectively at the third day, seventh day , 14th day , 21st day,28th day and every 35th day after immunization each group takes 4 chicknes randomly to detect antibody level and IFN-r concentration in seurm , T cell activity ratio and NO concentration in peripheral blood and indexes of immunity orgnas. Eighteenth day in the Immune scheduled time ,twenty chicknes of each group were used to challenge and proteetion test.The results indieated that rIL-18 could improve immune Protection for NDV vaccine and elevate antibody level and IFN-r concentration in seurm,T cell activity ratio and NO concentration in peripheral blood and indexes of immunity orgnas. And some of the difference was significant (0.01
In conclusion, the chicken IL-18 were successfuly expressed in E. coli BL21 prokaryotic cells and eukaryotic cells Pichia pastoris X-33 respectively,in this study.And all the rchicken IL-18 has the biological activity .They can improve immune Protection for NDV vaccine.
引文
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