猪IL-2基因真核表达载体的构建及在酵母中的表达

用真核表达引物从pGEM-IL-2重组质粒中扩增出猪IL-2基因，将目的基因和真核表达载体pPIC9K连接转入E．coli的JM109中，得到了猪pPIC9K-IL-2重组表达质粒。通过电激法将经SalⅠ酶切线性化的pPIC9K-IL-2质粒转化到巴斯德毕赤酵母GS115感受态细胞中，利用甲醇诱导表达，经SDS-PAGE电泳分析，表明在摇床水平及发酵罐中均表达出约17ku大小的分泌性目的蛋白，采用Sephadex G-100分子筛层析对其表达产物进行纯化，纯化结果理想。     

猪IL-2与IL-6的融合表达及活性研究

为获得具有猪白细胞介素-2(pIL-2)和猪白细胞介素-6(pIL-6)双重活性的融合蛋白,研究其作为高效免疫佐剂的可行性,本研究利用基因重组技术将克隆到的pIL-2和pIL-6的成熟肽基因利用一段柔性Linker序列串联后插入到原核表达载体pBV220中,转化大肠杆菌,42℃诱导表达得到融合蛋白pIL-6-2,对蛋白进行纯化复性后用MTT法检测其生物学活性。结果显示不同浓度pIL-6-2蛋白对小鼠脾淋巴细胞的增殖活性差异很大,0.1μg/mL浓度的pIL-6-2活性最好。本研究为利用该蛋白作为高效免疫制剂的应用奠定了良好基础。     

猪白细胞介素2基因的克隆和酵母表达

以伴刀豆球蛋白A（ConA）诱导的猪外周血淋巴细胞中提取的总RNA为模板,采用RT-PCR技术扩增出约500 bp的DNA片段,对阳性克隆进行测序与分析。结果：所克隆的基因与GenBank上公布的猪白细胞介素2（PoIL-2）基因的同源性为100%,表明试验成功获得了PoIL-2基因的全序列克隆;以该重组质粒为模板进行PCR,扩增出PoIL-2成熟蛋白的基因片段,连接真核表达载体pPICZαA,成功地构建了重组PoIL-2成熟蛋白基因的真核表达载体pPICZαA-PoIL-2;电转化pPICZαA-PoIL-2于巴斯德毕赤酵母X-33,诱导表达后进行表达产物的SDS-PAGE鉴定,结果表明试验成功地建立了重组PoIL-2的酵母表达系统。     

猪IL-18基因原核表达载体的构建及表达

以含新兴猪IL-18基因的真核质粒pcDNA3.1-pIL18为模板,扩增出新兴猪IL-18基因,将其定向克隆至原核表达载体pET32c和pET41c的SacⅠ和KpnⅠ位点,构建了原核表达载体pET32c-pIL18和pET41c-pIL18。经酶切和PCR鉴定,表明所构建的重组质粒为特异性新兴猪IL-18基因原核表达质粒。将该重组质粒转化大肠埃希菌BL21(DE3)细胞,筛选的阳性菌落经SDS-PAGE分析及Western-blotting分析,表明成功构建了表达重组猪IL-18的大肠埃希菌基因工程菌株。蛋白的可溶性分析表明,重组蛋白呈不可溶表达,在菌体内以不溶性的包涵体形式存在。     

猪IL-18基因的原核表达及重组蛋白的纯化

以pcDNA3．1-pIL-18为模板，采用PCR技术扩增到了猪白细胞介素18（IL-18）的成熟蛋白基因，通过KpnⅠ＋SacⅠ双酶切及连接反应，构建了pET32c—pIL—18原核表达质粒。经过限制性内切酶分析、PCR鉴定及DNA序列测定证实，重组质粒中的基因片段连接正确。之后，重组质粒转化大肠杆菌BL21（DE3），于37℃、1．0mmol／L IPTG条件下诱导表达。菌体裂解产物经SDS—PAGE分析，在分子质量约为33ku处出现了预期的目的蛋白。用8mol／L脲对表达产物变性，经Ni^2＋NTA柱纯化，透析复性，得到了纯化的IL-18蛋白。Western—blot分析证实，纯化的重组IL-18蛋白具有反应活性。上述研究结果为重组IL-18的应用奠定了基础。     

鸡IL-18酵母表达载体的构建及表达条件的优化

应用PCR技术从含有罗曼鸡白介素18全长基因质粒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,构建了重组质粒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,筛选多拷贝单克隆进行诱导表达,并进一步分析了培养液pH值、诱导剂浓度、诱导时间和诱导温度对重组菌表达水平的影响,从而获得最优化的表达条件。结果表明,重组毕赤酵母菌能够表达鸡IL-18,且在培养液pH6.0,甲醇诱导浓度1.5%,诱导温度26℃的条件下诱导96 h,目的蛋白的表达量最高,可占菌体总蛋白的60.2%。本文为鸡IL-18的规模化发酵生产奠定了基础。     

猪IL-2基因在毕赤酵母中的高效表达及生物活性分析

本研究根据密码子偏爱原则设计猪IL-2引物,克隆其基因后构建pPIC9k-IL-2重组表达载体,线性化后电转化毕赤酵母GS115,获得优化密码子的重组酵母转化子;再用G418抗性梯度法筛选得到多拷贝重组菌株,不同条件下进行甲醇诱导表达。结果表明:经PCR鉴定IL-2基因已整合到酵母基因组中。表达产物经SDS-PAGE电泳分析发现相对分子质量为16、20ku两条目的蛋白表达带,脱糖基化分析表明目的蛋白得到了适度的糖基化修饰,Western blotting分析显示表达的蛋白具有良好的免疫反应性,分子筛纯化可以获得纯度为95%的蛋白质,淋巴细胞增殖活性分析表明所得的蛋白质具有促淋巴细胞增殖的活性。毕赤酵母表达系统可以高效地表达具有生物活性的重组猪IL-2蛋白分子。     

羊白细胞介素-18成熟蛋白的酵母表达和生物学活性研究

为了研究重组羊白细胞介素(gIL)-18在酵母系统中的高效表达,进一步阐明该重组蛋白的生物学活性,以含有gIL-18基因的重组质粒为模板进行PCR扩增,构建重组表达质粒pPICZ-gIL-18,转化于毕赤酵母GSll5,以甲醇诱导表达,经sDS-PAGE和western blot分析证实了重组蛋白的表达,分泌的重组gIL-18表达量为100 mg/L.经纯化后用MTT法和MDBK-VSV法检测表达的重组IL-18体外生物活性,利用免疫试验检测了重组蛋白对羊痘疫苗的免疫增强作用.实验结果表明,该蛋白具有诱导MDBK细胞分泌IFN-γ和刺激PBMC增殖的生物学活性,比活性为1.5×105 u/mg.体外具有增强羊痘疫苗的活性.毕赤酵母分泌表达的重组gIL-18具有良好的生物学活性,为gIL-18作为免疫佐剂和免疫治疗剂的大规模应用奠定了基础.     

鸡IL-18成熟蛋白基因变构体毕赤酵母表达载体的构建及表达

为使鸡IL-18成熟蛋白基因在真核系统中高效表达,对其进行定点突变,将酵母低频密码子突变为偏嗜性密码子,构建了鸡IL-18成熟蛋白变构基因真核重组表达载体pPICZαA-MChIL18*,并将其转化入P.pastoris X-33.重组菌株X-33/pPICZαA-MChIL18*经甲醇诱导后,表达产物的上清液经SDS-PAGE检测,结果在约23 000处有目的条带,与预期的相对分子质量一致,占总蛋白的45%,对其进行Western-blot检测,结果与兔抗鸡IL-18多克隆抗体发生特异性免疫反应.结果表明,鸡IL-18成熟蛋白变构基因在毕赤酵母中成功的进行了表达.     

猪白细胞介素-2在甲醇酵母中的表达

将猪白细胞介素-2(pIL-2)的完整cDNA序列克隆到甲醇酵母(Pichiapastoris)表达载体pPIC3 5K中,在E.coli体系中鉴定得到阳性克隆子pPIC3 5K-IL2。将pPIC3 5K-IL2经SacI线性化后,转化入经LiC1致敏的P.PastorisGS115菌株中,得到的重组菌株经1%浓度甲醇诱导后,利用SDS-PAGE电泳,斑点杂交及去糖基化酶消化证实,在培养上清中获得了分泌型表达的猪白细胞介素-2(蛋白分子量约为20KD),其表达量可达到80mg/L。用CTLL-2细胞进行活性检测,生物学活性可达2×106IU/ml。对其表达情况进行时间梯度分析,确定第5天表达量达到最高点,为最佳诱导时间;分析连续4个批次的重组菌株表达情况,结果表明重组菌株在适当菌体浓度下连续培养均能稳定、高效的表达外源蛋白pIL-2。本实验为进一步大规模生产pIL-2提供了理论依据。     

猪白细胞介素-2在BHK-21细胞中表达及其活性分析

为鉴定BHK-21细胞中稳定表达猪白细胞介素-2(PoIL-2)的活性,本研究利用RT-PCR技术从猪的淋巴细胞中克隆PoIL-2基因,将其连接于pcDNA3.1载体中,并转染BHK-21细胞.经过G418筛选及RT-PCR鉴定,获得稳定表达PoIL-2基因的BHK-21细胞系.采用实时定量PCR及ELISA测定PoIL-2在BHK-21细胞内的表达,同时以淋巴细胞增殖试验(MTT)检测PoIL-2蛋白活性.试验结果表明稳定整合PoIL-2基因的BHK-21细胞系其PoIL-2 mRNA的转录效率是阴性对照组1.43倍,蛋白表达量是阴性对照组1.29倍.MTT试验表明真核表达的PoIL-2促淋巴细胞增殖活性与阴性对照组相比差异极显著(p＜0.01),表明BHK-21细胞表达的PoIL-2具有良好的生物学活性.本研究为开发新型基因工程疫苗佐剂和抗病毒制剂奠定了基础.     

猪IL-2成熟蛋白在昆虫细胞中的表达及生物学活性鉴定

以含猪IL-2基因的重组质粒pGEM-pIL2为模板,PCR扩增猪IL-2成熟蛋白基因。将猪IL-2成熟蛋白基因定向克隆到杆状病毒转移载体pFastBac Dual中,转化含穿梭载体Bacmid的感受态细胞DH10Bac,经抗性及蓝白斑筛选得到含猪IL-2基因的重组质粒rBacmid-IL2,转染昆虫细胞。间接免疫荧光试验表明重组猪IL-2在Sf9昆虫细胞中获得了表达,SDS-PAGE可检测到相对分子质量为20000的重组蛋白,Western-blot证实该重组蛋白可与兔抗猪IL-2单克隆抗体发生特异性反应,重组猪IL-2经纯化后能明显促进猪T淋巴细胞转化。结果表明,重组猪IL-2在昆虫细胞中得到表达,表达的重组蛋白具有一定生物学活性,为进一步开发研制新型免疫佐剂奠定了基础。     

Large-scale production of porcine mature interleukin-18 (IL-18) in silkworms using a hybrid baculovirus expression system

In this report, a hybrid baculovirus expression system, which means a hybrid virus of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of porcine mature interleukin-18 (IL-18) in silkworms. Two recombinant hybrid baculoviruses containing cDNA of the porcine precursor IL-18 and the porcine caspase-1 were constructed and were used to infect silkworm larvae. After the co-infection of the two viruses, porcine mature IL-18 was efficiently produced in the haemolymph. The concentration of IL-18 in the haemolymph was 80-100 microg/ml, as determined by porcine IL-18 specific ELISA. This yield was twenty-times more than that of the insect cell expression system described previously. The porcine mature IL-18 produced by the silkworms strongly induced interferon-gamma (IFN-gamma) production from porcine PBMC. An insect factory system for the large-scale production of useful cytokines for livestock animals will be available in the near future.     

First detection of porcine norovirus GII.18 in Latin America

Human (Hu) noroviruses (NoVs) circulate worldwide infecting people of all ages in developing and developed countries. Animal NoVs present some antigenic and genetic relationship to HuNoVs, although their zoonotic potential has not been established yet. Among animal NoVs, porcine (Po) NoVs are the most genetically related to HuNoVs. PoNoVs have only been detected in healthy finisher pigs in a few developed countries. Information about them lacks in developing countries. In this study 96 fecal samples from pigs of different ages from five farms in Rio de Janeiro State, Brazil were tested for NoVs. We report detection and genotyping by RT-PCR, nucleotide sequencing and phylogenetic analysis of partial polymerase and capsid regions of viral genome PoNoV genogroup II genotype 18 (GII.18) in one stool sample from a healthy finisher pig. This is the first report of PoNoV detection in Latin America and it supports the assumption that PoNoVs present a worldwide distribution.     

Expression of cytokeratin 18 during pre- and post-natal porcine lung development

The expression pattern of the intermediate filament protein cytokeratin 18 (CK 18) is described during pre- and post-natal development of the porcine lung using a monoclonal antibody against human CK 18. Lungs from 16 foetuses in pseudoglandular, canalicular, saccular and alveolar stages of lung development and lungs from 12 pigs ranging in age from birth to 49 days after birth were studied by immunohistochemistry.In the early pseudoglandular stage of development (day 70 of gestation) all the columnar epithelial cells lining the tubular endbuds strongly expressed CK 18 predominantly in the apical cell compartment. A modest staining was found in the more cuboidal cells of the canalicular stage (day 80 of gestation) where the labelling occurred as a distinct positive rim at the apical cell membrane in most of the cells lining the canaliculi. In 96- and 100-day-old foetuses, parts of the gas exchanging area were formed as terminal sacs by extreme attenuation of the epithelium. In this stage, CK 18 was clearly detectable in the flat type I as well as in the cuboidal type II alveolar epithelial cells. A marked change of the CK 18 expression pattern occurred during formation of the alveoli by septal outgrowth and maturation of the epithelium in 105- and 111-day-old foetuses. Differentiated type I cells no longer expressed CK 18, whereas type II cells were still labelled. Moreover, a specific change in the subcellular distribution pattern from the luminal periphery in immature porcine type II cells to a cytoplasmic localization in differentiated type II cells could be observed. Our investigation additionally demonstrated that the epithelium of bronchi, bronchioli and terminal bronchioli expressed CK 18 in all pre- and post-natal developmental stages. From the 96 days of gestation onwards the epithelial cells of developing bronchial glands were also labelled. Our results clearly show that during porcine lung development profound changes in the cellular expression pattern of CK 18 occur and that CK 18 can be regarded as a selective marker for differentiated porcine alveolar type II cells from the 105th day of gestation onwards. We also assume that the intermediate filament CK 18 could be of significance in the maturation process of the type II alveolar cells.     

猪圆环病毒2型感染对猪肺泡巨噬细胞生物学活性的影响

将猪圆环病毒2型（PCV2）BF株经口、鼻接种40日龄健康仔猪，在接种后不同时间宰杀，收集猪肺泡巨噬细胞（PAM），同时设立对照。用FITC—Annexin V／PI双染色流式细胞术和琼脂糖凝胶电泳检测PAM凋亡现象，通过EA花环试验测定Fc受体数目，通过吞噬鸡红细胞试验测定吞噬功能，分析PCV2感染对PAM生物学活性的影响。结果显示，在整个试验期内2种方法均没有检测到PAM凋亡，表明PCV2感染不会诱发PAM凋亡。PAM的Fc受体数和吞噬鸡红细胞数的变化规律一致，与对照组相比，两者数量在接种后第3d明显下降，第7d有所回升，之后基本恢复，表明PCV2感染后PAM吞噬和清除病原的功能出现短暂下降。     

奶牛白细胞介素-18的温控表达和活性分析

白细胞介素-18 是机体先天性和获得性免疫的重要调节因子,在慢性炎症、自体免疫性疾病、各种各样的癌变及众多传染病的发生过程中都有表达[1].     

Shotgun proteomic analysis of porcine colostrum and mature milk

The epitheliochorial nature of the porcine placenta prevents the transfer of maternal immunity. Therefore, ingestion of the colostrum immediately after birth is crucial for neonatal piglets to acquire passive immunity from the sow. We performed a shotgun proteomic analysis of porcine milk to reveal in detail the protein composition of porcine milk. On the basis of the Swiss‐Prot database, 113 and 118 proteins were identified in the porcine colostrum and mature milk, respectively, and 50 of these proteins were common to both samples. Some immune‐related proteins, including interleukin‐18 (IL‐18), were unique to the colostrum. The IL‐18 concentration in the colostrum and mature milk of four sows was measured to validate the proteomic analysis, and IL‐18 was only detected in the colostrum (191.0 ± 53.9 pg/mL) and not in mature milk. In addition, some proteins involved in primary defense, such as azurocidin, which has never been detected in any other mammal's milk, were also identified in the colostrum.