肠道病毒71型外壳蛋白VP3在Pichia.pastoris酵母中的表达

利用逆转录聚合酶链式反应(RT-PCR)的方法克隆肠道病毒71型SHZH98株外壳蛋白VP3基因,连接T载体,经序列测定后,构建酵母分泌型表达质粒pPIC9K/VP3,经序列测定证实-factor信号肽和VP3的序列和阅读框正确后,用SacI酶切使之线性化,电转化法将目的基因整合到宿主菌GS115基因组上;经转化子表型筛选和PCR分析鉴定后,甲醇诱导,筛选到5株高效表达VP3蛋白的工程菌株.ELISA实验表明:重组蛋白VP3具有较好的免疫原性.     

RACK1蛋白在毕赤酵母GS115中的表达研究

本文克隆得到了拟南芥和水稻的活化态C激酶1受体基因,AtRACK1/OsRACK1,用电转化的方法成功将AtRACK1/OsRACK1基因整合到毕赤酵母GS115的染色体上,经菌体培养和甲醇诱导后获得了有效分泌表达,表达产物存在于培养液上清中,表达蛋白经SDS-PAGE鉴定显示其分子量为36kD左右,在诱导72小时后AtRACK1/OsRACK1蛋白表达量最大,该结果为进一步纯化RACK1蛋白,获得植物RACK1蛋白抗体奠定了基础.     

肝癌差异表达基因HCUTAL2在酵母Pichia pastoris中的表达

利用正常肝组织和肝癌组织的mRNA,通过逆转录方法,将Cy3和Cy5二种荧光分别标记到2种组织的cDNA上,制备成cDNA探针,并与包含4096条各种人类基因的DNA表达谱芯片进行杂交及扫描,重复11次实验,通过计算机数据处理判定基因是否在上述2种组织中存在表达差异,从而鉴定参与肿瘤发生的基因,其中1类差异表达基因为CUTA的高度同源基因,该基因可能参与重金属离子在体的代谢,对该基因拟编码的蛋白质进行酵母表达。     

LZ—8基因的克隆及其在毕赤酵母（Pichia pastoris）中的表达

用PCR从灵芝（Gamoderma lucidum)基因组扩增免疫蛋白LZ－8基因，将其构建到酵母表达质粒pPICZaA中，采用电激转化法转化毕赤酵母，获得转化子。对其Sourthern杂交分析，结果表明LZ－8基因整合到毕赤酵母基因组。对转化酵母作发酵培养，SDS－聚炳烯酰胺凝胶电泳检测到LZ－8蛋白的表达。     

重组人骨形态发生蛋白4在毕赤酵母中的表达研究

试图建立人骨形态发生蛋白-4在毕赤酵母(Pichia pastoris) 中的表达技术.包括:将hBMP4成熟片段从全长cDNA亚克隆到分泌型表达载体质粒pPIC9K多克隆位点上,构建能表达rhBMP4的载体质粒pPIC9K-hBmp4.经转化Pichia pastoris宿主菌株GS115,PCR鉴定基因整合后,摇瓶培养,用甲醇诱导表达.表达上清液经SDS-PAGE和Western-blot 分析,结果表明rhBMP4以单体形式分泌到发酵上清液中,单体分子质量大小约为26 ku,摇瓶表达量达到20.13 μg/mL.     

Monellin基因在酵母中的表达研究

本实验根据毕赤酵母偏爱密码子,人工合成了高甜度monellin基因,并构建了重组分泌型酵母表达载体pPIC9KM,通过电击转化获得了可高效分泌表达有甜味活性高甜度monellin的重组毕赤酵母GS115/pPIC9M.通过PCR检测证实monellin基因已经整合进酵母基因组,SDS-PAGE和Westerm blot免疫杂交知所表达的蛋白是目的蛋白,最后通过双盲测定证实表达的蛋白比同等重量的蔗糖甜约1000倍.     

人骨唾液酸蛋白在巴斯德毕赤酵母中的高效分泌表达

以PCR方法扩增人bsp基因，与分泌型表达载体pPICZαA重组，重组质粒经电击转化毕赤酵母GS115菌株，获得的GS115／pPICZαA－hbsp表达菌株能高效分泌表达rhBSP.SDS-PAGE和Western blot分析结果表明，产物的分子质量接近66ku，能发生特异性抗原－抗体反应。     

肿瘤抑制因子p53蛋白在毕赤酵母中的克隆表达

TP53基因在调控细胞信号通路和抑制肿瘤细胞中发挥着重要作用.本研究利用毕赤酵母表达系统以获得大量重组p53蛋白,为此,本文将人TP53基因克隆至表达载体p PIC3.5K,电转化进入毕赤酵母GS115,通过组氨酸筛选和G418筛选,获得高拷贝稳定转化子.SDS-PAGE和Western Blot检测结果表明p53蛋白可以在毕赤酵母中表达.本研究所获酵母菌株为今后大规模生产重组p53蛋白奠定了基础.     

外源蛋白在巴氏毕赤酵母中高效表达的策略

高效表达外源蛋白, 在理论和实践上特别是在生物制药中具有重要意义, 巴氏毕赤酵母( Pichia pastoris) 是表达外源蛋白最理想的真核表达系统之一.影响外源蛋白在 P. pastoris中表达的因素很多, 主要包括外源基因自身的特性、载体、宿主细胞几个方面,了解和灵活运用它们的联系,有助于获得外源基因在 P. pastoris 中的高效表达.     

基因工程菌Pichia pastoris高密度发酵表达重组人血清白蛋白

在摇瓶条件下对基因工程菌Pichia pastoris的发酵条件进行了实验，并根据摇瓶发酵的优化结果进行了补料分批高密度发酵。在分批发酵时，接种量为10%且种子细胞光密度（OD600）为20左右时，细胞生长的延迟期约为2.11h，细胞生长光密度与培养时间的关系模型为：y=0.7841e^0.2319t（线性相关系数r=0.9936）；在补料发酵时细胞浓度可达115g/L-160g/L（干重），在120h重组人血清白蛋白表达量达3.6g/L。     

Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope, and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity. GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope. All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies, 9D8 and 2D7, could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays. In the flow cytometry analysis, the mAbs 9D8 and 2D7 could bind to HIV-Env CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide, which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane. However, in syncytium assays, none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion. The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes. The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.     

Molecular motions and conformational transition between different conformational states of HIV-1 gp 120 envelope glycoprotein

The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and coreceptor CCR5 or CXCR4 on the surface of host cells to initiate virus entry. Both the crystal structures of the HIV-1 gp120 core bound by the CD4 and antigen 17b and the SIV gp120 core pre-bound by CD4 are known. Despite the wealth of knowledge on these static snapshots of molecular conformations,the details of molecular motions involved in conformational transition that are crucial to intervention remain elusive. We presented comprehensive comparative analyses of the dynamics behaviors of the gp120 in its CD4-complexed,CD4-free and CD4-unliganded states based on the homology models with modeled V3 and V4 loops by means of CONCOORD computer simulation to generate ensembles of feasible protein structures that were sub-sequently analysed by essential dynamics analyses to identify preferred concerted motions. The re-vealed collective fluctuations are dominated by complex modes of combinational motions of the rota-tion/twisting,flexing/closure,and shortness/elongation between or within the inner,outer,and bridg-ing-sheet domains,and these modes are related to the CD4 association and HIV neutralization avoid-ance. Further essential subspace overlap analyses were performed to quantitatively distinguish the preference for conformational transitions between the three states,revealing that the unliganded gp120 has a greater potential to translate its conformation into the conformational state adopted by the CD4-complexed gp120 than by the CD4-free gp120,whereas the CD4-free gp120 has a greater potential to translate its conformation into the unliganded state than the CD4-complexed gp120 does. These dynamics data of gp120 in its different conformations are helpful in understanding the relationship between the molecular motion/conformational transition and the function of gp120,and in gp120-structure-based subunit vaccine design.     

A conserved structural motif shared by EIAV gp90 and HIV gp120

Both HIV (human immunodeficiency virus) and EIAV (equine infectious anemia virus) belong to the retroviridae family and lentivirus genus. EIAV is similar to HIV not only in its morphology, genomic structure and antigenicity, but also in its replicative and infectious characteristics. Their env genes comprise surface unit (SU) and trans- membrane protein (TM). The large number of disulfide bonds and N-glycosylation sites are prominent structural properties of SU (HIV gp120 and EIAV g…     

大鼠组织型金属蛋白酶抑制剂-1在毕赤酵母中的重组表达

提取SD大鼠肝组织总RNA,通过RT-PCR扩增TIMP-1 cDNA片段.将扩增产物克隆至pUCm-T载体上,PCR、限制性酶切及测序证实后,EcoRI和NotI双酶切pUCm-T-TIMP-1重组体,回收TIMP-1,再将其亚克隆到酵母表达载体pPIC9K中,并将阳性克隆进行PCR、酶切和测序分析,构建pPIC9K-TIMP-1表达载体,电击转化到毕赤酵母,通过表型筛选和诱导表达得到蛋白表达工程菌,为进一步研究TIMP-1功能和作用机理等的研究创造了条件.     

抗HIV-1gp41合成多肽gp41-5单克隆抗体的制备及初步鉴定

制备抗HIV-1 gp41合成多肽gp41-5的单克隆抗体（mAb）,为筛选抗HIV-1多肽及分析gp41的抗原表位提供有用工具。常规动物免疫、细胞融合、克隆化制备抗gp41-5多肽mAb,并用ELISA法对其特异性、抗原识别表位及相对亲和力等做了初步鉴定。获得了4株抗gp41-5多肽的mAb,这4株mAb均特异识别gp41-5多肽,但不与gp41的N36或C34多肽片段反应。得到的4株mAb能特异结合gp41核心结构的空间构象。     

Molecular architecture of native HIV-1 gp120 trimers

The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection. Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with three-dimensional image classification and averaging, we report the three-dimensional structures of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells.     

纳豆激酶基因在巴斯德毕赤酵母中的表达

构建pPICZαA-NK重组质粒，并转化入E.coli TOP-10中，得到转纳豆激酶基因工程菌，提取重组质粒经单酶切，双酶切，PCR分析及序列测定，证明克隆到载体pPICZαA上的外源基因即为纳豆激酶基因，将重组质粒pPICZαA-NK线性化后，分别转化毕赤酵母GS115与KM71，在含Zeocin^TM的选择性YEPD平板筛选到重组酵母，经检测重组酵母发酵上清液中具纳豆激酶溶纤活性，SDS-PAGE电泳结果表明外源蛋白的表达量占菌体蛋白的10%左右。     

肠道病毒71型SHZH03株VP1基因在毕赤酵母中的表达

利用逆转录聚合酶链式反应(RT-PCR)方法扩增得到肠道病毒71型(EV71SHZH03)外壳蛋白VP1基因,经序列测定证实后,构建重组表达质粒VP1/pPIC9K,转化Pichia pastoris 酵母宿主菌Gs115,以Myc-Tag多克隆抗体作为一抗,利用双层滤膜法筛选酵母转化子.甲醇诱导表达.SDS-PAGE分析显示:表达产物的分子量约为30000,与天然VP1大小一致,ELISA实验表明,表达上清液可与EV71患者急性感染期血清呈阳性反应,表明重组蛋白VP1具有免疫原性.     

尿激酶体外抑制人体免疫缺损病毒的侵染能力

人体免疫缺损病毒的包膜蛋白gp120的V3环区包含一段在人类蛋白质中很少出现的高度保守序列，但这段序列与纤溶酶原被纤溶酶原激活剂酶切位点附近序列有同源性．由于V3环区在人体免疫缺损病毒侵染细胞过程中的重要性，评估了尿激酶对人体免疫缺损病毒侵染能力的影响．通过检测逆转录酶活力，P24抗原的表达和合胞体形成情况发现尿激酶可以抑制人体免疫缺损病毒对多种淋巴瘤和白血病细胞系，如MT4、CCM、H9和外周血单核细胞的侵染能力，并且这种抑制与尿激酶浓度呈剂量依赖关系．那些能够被尿激酶抑制的人体免疫缺损病毒株其V3环区序列必须与纤溶酶原激活区亭列同源，实验事常用病毒株包括BRU和RF以及某些野生病毒株．研究结果显示尿激酶在体外实验中可以抑制人体免疫缺损病毒的侵染能力．