人胰岛素在甲醇酵母Pichia pastoris中的分泌表达

将猪胰岛素前体基因和在其５’端引入９个氨基酸的间隔肽序列的ＰＩＰ基因插入到Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ的分泌表达质粒ｐＰＩＣ９中,得到分泌表达质粒ｐｐＩＣ９／ＰＩＰ和ｐＰＣ９／ｓｐ－ＰＩＰ７并用以转化ｐａｓｔｏｒｉｓ ＧＳ１１５。用点杂交筛选,获得高拷贝转化Ｐ３９（－ｓｐ）和Ｓ５１（＋ｓｐ）。     

人胰岛素原在甲醇酵母(Pichia pastoris)中的高效表达

甲醇营养型酵母Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ在近十几年已被人们广泛用作外源基因表达的系统。表达的是可溶性蛋白,且胞外分泌。本研究系将外源基因（胰岛素原基因）连接到穿梭质粒ＰＨＩＬ－Ｓ１上,再通过同源重组到酵母染色体,筛选表达株。用１ 升发酵罐在甲醇诱导下可获得０．３ｇ／Ｌ胰岛素原的产率。     

猪白细胞介素-6在巴斯德毕氏酵母(Pichia pastoris)中的分泌表达

白细胞介素6 (Interleukin_6 ,IL_6 )是一种具有多种生物学效应的细胞因子,在疾病诊断与疫苗佐剂领域有广阔的应用前景。在本试验中,猪白细胞介素_6 (pIL_6 )的cDNA序列被克隆入甲醇酵母(Pichiapastoris)分泌表达载体pPIC9K中,并转化入P .pastorisGS115菌株。其重组菌株GS115 pPIC9K_IL6经1%甲醇诱导后,能分泌表达分子量约为2 4 5KD的重组蛋白,Westernblot确证为pIL_6。该酵母表达产物无N端糖基化修饰。用依赖IL6生长的B9细胞株检测提纯后的pIL_6 ,其生物学活性可达8×10 4 IU mg。     

结核分枝杆菌ESAT-6蛋白在毕赤酵母中的融合表达及其活性研究

为了获得结核分枝杆菌ESAT 6蛋白在毕赤氏酵母中的高效表达 ,将人α-2a干扰素基因、结核分枝杆菌esat-6基因经PCR扩增并加上相应的酶切位点 ,两基因之间的DNA接头编码肠激酶识别的多肽 ,DNA经酶切连接插入分泌型载体pPIC9K ,将重组表达质粒pPIC9K-α2a-esat6用SalⅠ单酶切之后 ,电击转入PichiapastorisSMD1168中 ,采用G418梯度筛选获得高抗性转化子。以甲醇作为诱导物 ,发酵4d后取上清 ,进行SDS PAGE和Westernblot鉴定并分析表达产物的干扰素活性。结果表明 ,重组菌株成功地分泌表达了分子量大小约为 30kD的融合蛋白 ,表达产物不仅具有较高的干扰素活性 ,而且可以和结核病人血清发生特异性结合 ,为结核病的特异性诊断和结核病新型疫苗的研制打下了基础.     

鼠卵透明带3在Pichia pastoris酵母中的分泌表达

本研究通过酵母Pichiapastoris表达鼠卵透明带 3(mZP3)蛋白 ,获得控制害鼠生育研究的抗原物质。根据国际基因库已发表的mZP3基因序列设计引物 ,并分别在 5’引物和 3’引物中引入EcoRI和XbaI酶切位点。经PCR扩增 ,将mZP3克隆至穿梭质粒pGAPZαA上 ,获得的重组穿梭质粒pGAPZαA mZP3经线性化后 ,采用LiCl法转入毕赤酵母SMD1 1 68菌株中 ,Zeocin+ 筛选阳性克隆 ,酵母发酵上清液进行SDS PAGE和Western blot检测 ,结果表明mZP3在酵母中得到了特异性表达 ,表达产物的分子量约为 60kDa ,说明mZP3能够在酵母真核系统中成功表达 ,并可以用作mZP3免疫不育控制鼠害的检测抗原。     

HPV16 E6蛋白在宫颈癌中的作用研究进展

高危型人乳头状瘤病毒16型(HPV16)与50%以上的宫颈癌密切相关,其E6癌蛋白作为病毒生命周期的主要蛋白之一,在诱导肿瘤发生与发展进程中起重要作用,且与病毒复制、宿主细胞周期调控、细胞凋亡、细胞增殖、细胞恶性表型转化有关。E6蛋白主要作用包括:通过结合E6相关蛋白降解P53抑制细胞凋亡;增强端粒酶活性使宿主细胞永生化;与Daxx启动子区结合,抑制启动子转录活性,降低Daxx蛋白表达,阻遏细胞凋亡;与多种细胞因子相互作用后,经多种途径改变细胞微环境,使之有利于肿瘤细胞逃避宿主固有免疫应答。因此,在宫颈癌的发生和发展中,HPV16 E6蛋白通过多种作用机制发挥重要作用。     

登革2型病毒E蛋白在酵母菌中的分泌表达

以 pPICZαB为载体 ,应用RT PCR从感染D2V的C6 / 36病变细胞中克隆全长E基因 ,电转化法将重组质粒整合入巴斯德毕赤氏酵母菌 ,经抗生素筛选、表型鉴定和PCR分析得到Mut 型的多拷贝整合菌 ,经甲醇诱导培养可产生 6 9kD的融合蛋白 ,与含组氨酸尾的D2V包膜糖蛋白分子量理论值相符 ;免疫印迹证实该表达产物可与D2VE特异性单抗和D2V多抗进行反应 ;表达产物经金属螯合亲和层析可获得纯化的含组氨酸尾的E融合蛋白并保留其免疫反应性。研究显示克隆的全长D2VE基因可在毕赤氏酵母菌中高效分泌表达 ,E融合蛋白最大表达量 0 .1g/L。     

登革2型病毒E蛋白在酵母菌中的分泌表达

以pPICZ α B为载体,应用RT-PCR从感染D2V的C6/36病变细胞中克隆全长E基因,电转 化法将重组质粒整合入巴斯德毕赤氏酵母菌,经抗生素筛选、表型鉴定和PCR分析得到Mut+型的多拷贝整合菌,经甲醇诱导培养可产生69kD的融合蛋白,与含组氨酸尾的D2V包膜糖 蛋白分子量理论值相符;免疫印迹证实该表达产物可与D2V E特异性单抗和D2V多抗进行反应; 表达产物经金属螯合亲和层析可获得纯化的含组氨酸尾的E融合蛋白并保留其免疫反应性. 研究显示克隆的全长D2V E基因可在毕赤氏酵母菌中高效分泌表达,E融合蛋白最大表达量0.1g/L.     

人工设计的Ribozymes体外切割HPV16 E6和E7 mRNA的研究

人乳头状瘤病毒(human papillomavirus,HPV)是一类感染人体的皮肤粘膜并引起乳头状瘤病变的病毒。目前发现的HPV已有63型,不同型的HPV引起人体不同部位的病变。HPV 16主要与生殖系统的肿瘤尤其是子宫颈癌有密切关系。近年来的实验表明     

HPV16 E6对HeLa细胞中Rap1GAP蛋白水平下调的研究

目的探讨HPV16E6对细胞中Rap1GAP蛋白水平的影响,为阐明宫颈癌发生发展的分子机制提供实验依据。本研究组前期实验显示,宫颈癌石蜡切片组织中Rap1GAP蛋白水平下降,且与高危型HPV16/18感染相关。本文将进一步探讨HPV16 E6是否导致Rap1GAP蛋白下调的原因。方法通过将HPV16 E6基因插入pGEX-KG的BamHI和Hind Ⅲ酶切位点构建GST标记的HPVE6质粒,采用脂质体转染法将其转染入HeLa细胞中,Westernblot方法观察GST-tagged-HPV16 E6在HeLa细胞中的表达,并观察其对HeLa细胞中内源性Rap1GAP蛋白水平的影响。结果测序表明成功构建GST标记的HPV16E6质粒;Western blot检测表明HPV16E6在HeLa细胞中成功表达;并且发现HeLa细胞中过表达HPV16 E6后,Rap1GAP蛋白相对含量(0.602±0.205)明显低于未转染组(1.130±0.163),差异具有统计学意义(P0.05)。结论HPV16 E6下调Rap1GAP蛋白水平。     

High-level expression and characterization of a secreted recombinant cation-dependent mannose 6-phosphate receptor in Pichia pastoris

Mannose 6-phosphate receptors (MPRs) form essential components of the lysosomal enzyme targeting system by binding newly synthesized acid hydrolases with high (nM) affinity. We report the use of Pichia pastoris as a host to efficiently express the extracytoplasmic ligand-binding domain of the cation-dependent mannose 6-phosphate receptor. A truncated and glycosylation-deficient form of the receptor AF-Asn(81)/Stop(155) was secreted into the culture medium, yielding approximately 28mg/L after purification, which is an improvement of 10-100-fold compared to expression in baculovirus-infected insect cells and mammalian cells, respectively. Enzymatic deglycosylation indicated high-mannose sugars at the single potential glycosylation site of Asn 81. The extent and heterogeneity of N-glycans were revealed by applying matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the case of AF-Asn(81)/Stop(155), the majority (75%) of the oligosaccharides contained chain lengths of Man(8-10)GlcNAc(2) while Man(11-12)GlcNAc(2) comprised the remaining (25%) N-linked sugars. A comparative MALDI-TOF spectra of Asn(81)/Stop(155) purified from insect cells indicated that Man(2-3)GlcNAc(2) and GlcNAcMan(2-3)GlcNAc(2) share the oligosaccharide pool. The receptor isolated from yeast was functional with respect to ligand binding and acid-dependent dissociation properties, as determined by pentamannosyl phosphate-agarose affinity chromatography. In addition, the protein was biochemically and functionally similar to Asn(81)/Stop(155) expressed in insect cells concerning its oligomeric state and binding affinity to the lysosomal enzyme, beta-glucuronidase (K(d)=1.4nM). These results demonstrate that P. pastoris is a convenient system for the production of large quantities of functional recombinant MPRs suitable for structure-function studies.     

Human Papillomavirus (HPV) 16 E6 seropositivity is elevated in subjects with oral HPV16 infection

IntroductionHuman Papillomavirus (HPV) 16 E6 serum antibodies are common in people with HPV-related oropharyngeal cancers (HPV-OPC), but not the general population. We explored HPV16 seroprevalence in people with and without oral HPV16 infection, the cause of HPV-OPC.MethodsOral rinse samples were collected semiannually and tested for 36 types of HPV DNA by PCR. HPV16 E6 serum antibodies were tested at the visit of first oral HPV detection in participants with prevalent (n = 54), or incident (n = 39) oral HPV16 DNA; or at baseline in matched participants with no oral HPV16 DNA (n = 155) using multiplex serology assay. Predictors of seropositivity were examined using logistic regression.ResultsHPV16 E6 seropositivity (7.5% vs 0.7%; p = 0.005) but not seropositivity to the other HPV16 antigens, was significantly more common in those with than without oral HPV16 infection. There were only 8 HPV16 E6 seropositive participants, but oral HPV16 DNA remained a strong predictor of E6 seropositivity after adjustment for other risk factors (aOR = 14.6 95%CI, 1.7–122.5). Seroprevalence was similar in those with prevalent (7.4%; 4/54), and incident (7.7%; 3/39) oral HPV16 infection (p = 1.00). E6 seroprevalence was associated with reduced oral HPV16 clearance, but was not statistically significant (HR = 0.65 95% CI, 0.16–2.70).Seropositive participants were primarily male (87.5%), HIV-positive (75.0%; median CD4 cell-count of 840) and had oral HPV16 DNA (87.5%). History of an HPV-related cancer (0/8) or HPV-related anogenital dysplasia (1/8) was rare, and 4 participants had recent screening showing no anogenital dysplasia.DiscussionHPV16 E6 seropositivity was higher among people with than without oral HPV16 infection, despite no known anogenital disease in these participants.     

Functional properties of glycosylated lysozyme secreted in Pichia pastoris

Various mutant lysozymes having the N-glycosylation signal sequence, R21T (Asn(19)-Tyr(20)-Thr(21)), G49N (Asn(49)- Ser(50)-Thr(51)), R21T/G49N (Asn(19)-Tyr(20)-Thr(21)/Asn(49)-Ser(50)-Thr(51)), were secreted in the Pichia pastoris expression system. The secreted amounts of these mutant glycosylated lysozymes were almost the same as those of wild-type lysozyme (about 30 mg/liter). Glycosylation of the mutant lysozymes was confirmed by SDS-PAGE patterns, Endo-H treatment, TOF-MS analysis and chemical analysis. The composition of the carbohydrate chain attached to the single glycosylated lysozymes, R21T and G49N, was GlcNAc(2)Man(9-11), while that of the double glycosylated lysozyme, R21T/G49N, was GlcNAc(4)Man(27-32). The results of a CD analysis and lytic activity suggested that the conformation of the single glycosylated lysozymes had been conserved, while that of the double glycosylated lysozyme was less stable. The emulsifying properties of the lysozyme when glycosylated were greatly improved, being especially noteworthy in the double glycosylated lysozyme.     

毕赤酵母菌高效表达乙肝表面抗原的研究

目的:筛选高效表达HBsAg的毕赤酵母茵,制备目的蛋白.方法:从已确诊的乙肝病人血清中提取DNA,PCR扩增HBVS基因,将其分别克隆入毕赤酵母胞内表达栽体pPICZA中.构建重组质粒pPICZA-S和pPICZA-SH,经Sac I线性化后,LiCI化学法转化入酵母茵株GS115、X-33、KM71H和SMD1168.结果:诱导表达后的GS115工程茼单位体积的培养基所得的抗原含量最高,诱导培养基中加入0.1%酪蛋氨基酸后,可抑制目的蛋白的水解,有利于目的蛋白的表达,粗略估算表达量为15.3mg/L,最佳收获时间为72 h.结论:经SDS-PAGE和Westcrn-blot分析表明,所得产物为乙肝表面抗原S蛋白.     

新疆维吾尔族妇女宫颈癌组织中乳头瘤病毒16型E6基因的克隆和序列分析

为了分析新疆南部地区维吾尔族妇女宫颈癌组织中HPV16型E6基因结构特点，从中国新疆南部地区维吾尔族妇女宫颈癌活检组织标本中提取DNA，以宫颈癌活检组织标本DNA为模板进行PCR扩增，获得HPV16 E6基因，将其克隆到pUCm-T载体上，并对其进行基因全序列分析.PCR检测结果显示宫颈癌组织中HPV16 E6阳性率为82.35%（14/17）；测序结果显示，新疆株HPV16 E6基因全长456 bp，大小与德国标准株一致.E6基因的第247位碱基发生T→G突变，并由此引起所编码的氨基酸亦发生改变.上述结果表明，中国新疆南部地区维吾尔族妇女宫颈癌患者组织中HPV16 E6的基因结构与德国标准株HPV16 E6基因之间存在差异.     

Gene expression in yeast: Pichia pastoris.

Recent studies have shown the versatility and utility of the Pichia pastoris expression system. Improvements in strains have boosted the yield of proteins and peptides to the commercially feasible range. The Pichia pastoris expression system will soon be used to manufacture proteins for human clinical trials.     

蜡样芽孢杆菌胶原酶基因colR75E在毕赤酵母中的重组表达

【目的】利用毕赤酵母真核表达系统表达蜡样芽孢杆菌胶原酶Col R75E,寻找一种安全、稳定的方式体外制备具有高活性的胶原酶。【方法】以蜡样芽孢杆菌R75E基因组DNA为模板,采用PCR法扩增胶原酶col R75E基因,构建p PICZαA/col R75E重组质粒,将该质粒线性化后电转化至毕赤酵母X-33菌株,诱导其表达并对表达条件进行优化。将表达后的酵母发酵液上清通过硫酸铵沉淀、脱盐处理及亲和层析纯化步骤获得高纯度重组Col R75E胶原酶。利用胶原酶活力测定、SDS-PAGE电泳、胶原酶谱、I型胶原蛋白及不同底物蛋白降解产物电泳等方法对重组胶原酶Col R75E的活性及底物特异性进行分析。【结果】毕赤酵母中最佳表达重组胶原酶Col R75E的条件为p H 6.0,甲醇终浓度为2.5%,诱导时间72 h,诱导后的蛋白经SDS-PAGE、胶原酶谱以及I型胶原蛋白降解产物电泳分析发现,毕赤酵母中表达的重组胶原酶分子量符合预期,蛋白纯度超过95%,具有较好的胶原蛋白水解活性并测得其比活力为4.977 U/mg。该酶对I型胶原蛋白表现出较好的专一性,但是对牛血清白蛋白、酪蛋白及溶菌酶蛋白没有水解活性。【结论】利用毕赤酵母真核表达系统能够获得高活性的蜡样芽孢杆菌胶原酶Col R75E,为该胶原酶广泛应用于医疗、食品等工业领域奠定了理论和方法基础。     

Recombinant protein expression in Pichia pastoris

The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.     

巴斯德毕赤酵母表达系统研究进展

巴斯德毕赤酵母表达系统现在已经发展成为一种高效的外源蛋白基因优秀表达系统,该系统具有高表达、高稳定、高分泌、容易放大和成本低等优点,目前已有多种外源蛋白基因在该系统中实现高效表达,对巴斯德毕赤酵母表达系统的进一步研究将会促进其大规模的工业化应用。     

Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris

High-level recombinant expression systems for the production of stable triple-helical human collagens and collagen fragments have been developed in the yeast Pichia pastoris. Collagen fragments are secreted as single-chain polypeptides by the yeast alpha-mating factor pre-pro sequence, but secretion of full-length triple-helical procollagen molecules has not been achieved despite the use of the same secretory signal. We studied here the effects of the secretory signal and the conformation and size of the collagen polypeptide on its secretion in P. pastoris. Unlike the collagen signal sequence, the alpha-mating factor pre-pro sequence led to efficient secretion of single-chain 45 and 9 kDa type I collagen fragments. The efficiency was dependent on the length of the collagen polypeptide, as secretion of single-chain full-length 90 kDa alpha1(I) polypeptides was less efficient than that of the 45 kDa fragment. Furthermore, the conformation of the collagen polypeptides had a marked effect on secretion, as induction of trimerization of the 45 and 9 kDa fragments by either the C propeptide or the small trimerizing domain foldon led to an accumulation of triple-helical molecules inside the cells despite the presence of the alpha-mating factor pre-pro sequence. Our results show that P. pastoris is a suitable host for the development of tailored expression systems aimed at selective production of nonsecreted triple-helical and secreted single-chain collagen fragments of varying lengths for specific purposes.