多孔微球固定化CHO工程细胞生产rt-PA的研究

以 Ｃｙｔｏｐｏｒｅ 多孔微球固定产重组组织型纤溶酶原激活剂（ｒｔ Ｐ Ａ） Ｃ Ｈ Ｏ 工程细胞株４ Ｂ３ ,在２ Ｌ 搅拌式生物反应器用无血清培养基 Ｄ Ｆ５ Ｓ 连续灌流培养。４ Ｂ３ 细胞的最大活细胞密度和ｒｔ Ｐ Ａ 生产水平分别达到８８３ ×１０６／ ｍ Ｌ 和１２４７３ Ｉ Ｕ／ ｍ Ｌ。含ｒｔ Ｐ Ａ 的４ Ｂ３ 细胞培养上清经 Ｍ Ｐ Ｇ 吸附层析和 Ｌｙｓｉｎｅｓｅｐｈａｒｏｓｅ ４ Ｂ 亲和层析两步纯化,ｒｔ Ｐ Ａ 的纯度达到９８ ％ 。     

SDS－聚丙烯酰胺凝胶电泳纤维蛋白自显影法检测不同分子量的纤溶酶原激活剂

ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）纤维蛋白显影方法是经ＳＤＳ－ＰＡＧＥ将不同分子量的纤溶酶原激活剂（ＰＡ）分开,然后再转溶纤维蛋白板使之产生溶带而检测ＰＡ物质活性及分子大小的方法。此法与Ｗｅｓｔｅｒｎ印迹方法比具有检测更简便、灵敏、快速的特点,且是活性检测,但其分子量分析精度不如Ｗｅｓｔｅｒｎ印迹法。本研究用ＳＤＳ－ＰＡＧＥ纤维蛋白自显影方法对本室表达的重组组织型－ｐＡ（ｒｔ－ＰＡ）及突变体Ｎｌｌ７Ｑ／Ｎｌ８４Ｑ／△（Ｋ２９６——Ｇ３０２）及标准尿激酶（ＵＫ）和ｒｔ－ＰＡ进行了分析鉴定。     

高压快速柱分离纯化牛血球超氧化物歧化酶的研究

以牛血球为材料,经溶血等处理和丙酮沉淀,获得牛血球超氧化物歧化酶粗品。此粗酶可以通过ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ和ＣＭ－Ｓｅｐｈａｒｏｓｅ快速柱层析,获得超氧化物歧化酶纯品。纯化的酶比活可达１３５００ｕ／ｍｇ,经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和快速蛋白液相色谱（ＦＰＬＣ）检测,结果表明,纯化酶是均一的Ｓｅｐｈａｄｅｘ Ｇ－１００凝胶过滤测得该酶分子量为３１,８００,ＳＤＳ－ＰＡＧＥ测得亚基分子量为１５     

猪精子中与卵透明带糖蛋白ZP3结合的蛋白质

依次经ＰＳＬ－Ｓｅｐｈａｒｏｓｅ亲和层析柱和纤维素ＣＭ－５２离子交换层析柱,从猪精子的ＣＨＡＰＳ抽提液分离得４个蛋白质组分。用固相透明带精蛋白结合试验（ＩＺＰＧＢＡ）检测;表明精子蛋白ＳＰ１和ＳＰ２具有结合透明带糖蛋白ＺＰ３的活性,ＳＰ２并显示凝集血球的活性。精子蛋白ＳＰ１与卵预温育明显抑制精卵结合,抑制活性与加入的精子蛋白的浓度呈正相关。用生物素标记的ＺＰ３和蛋白质印迹技术,证明ＳＰ１中的６８ｋＤ精子蛋白与ＺＰ３结合,提示６８ｋＤ精子蛋白参与精卵结合。     

表面活性物质相关蛋白表达的调控研究进展

特异性的肺表面活性物质相关蛋白（ＳＰ）包括亲水性的ＳＰ－Ａ、ＳＰ－Ｄ和疏水性的ＳＰ－Ｂ、ＳＰ－Ｃ．它们的表达与合成受众多生理、病理因素影响。本文综述了该领域的研究进展。１．ＳＰ表达的组织细胞特异性调控：只有肺内某些细胞（肺泡Ⅱ型细胞、Ｃｌａｒａ细胞等）能合成分泌ＳＰ,这可能是由ＳＰ基因中特定序列决定的,如ＳＰ－Ｂ基因的细胞特异性表达的调控成分。２．ＳＰ表达的发育期调控：ＳＰ基因属发育控制基因家族。在人类妊娠前３ｍｏｎ胎肺中,ＳＰ基本不表达：妊娠１５－１８ｗｋ时,气管、支气管上皮细胞即可见ＳＰ－Ｂ、ＳＰ－ＣｍＲＮＡｓ和表达蛋白,它们可能比ＳＰ－Ａ出现早：妊娠１９－２０ｗｋ时可见ＳＰ－ＡｍＲＮＡ和表达蛋白,胎肺组织在体外无激素条件下培养可很快诱导ＳＰ表达,在妊娠后３ｍｏｎ内,各ＳＰｍＲＮＡｓ及其表达蛋白水平与磷脂水平平行升高,也与ＳＰ降低表面张力的特性逐渐增强相关,羊水中可检出这些蛋白,板层体的出现与ＳＰ－Ｂ的表达密切相关,而比ＳＰ－Ａ的表达早,胎肺发育过程中ＳＰｍＲＮＡｓ增加至少部分是由于其基因转录率升高,可能同时也与翻译增加有关。３．糖皮质激素对ＳＰ表达的调控：糖皮质激素对ＳＰ－Ａ表达的调控极复杂,且与剂量     

球形幽门螺杆菌分子生物学研究

为研究幽门螺杆菌（ＨＰ）球形变异本质,作者通过延期培养和采用亚抑菌浓度抗生素,使３株ＨＰ发生球形变异,对弯曲形和球形ＨＰ作了ＳＤＳ－ＰＡＧＥ、免疫印迹及４个毒力基因片段ＰＣＲ和ＰＣＲ－ＳＳＣＰ分析。ＳＤＳ－ＰＡＧＥ图谱显示球形ＨＰ分子量在７４×１０４以上的蛋白含量减少,免疫印迹显示球形ＨＰ１２５×１０４蛋白条带反应减弱,而抗生素诱变的球形ＨＰ分子量为１１×１０４和６３×１０４的蛋白条带反应增强。ＰＣＲ及ＰＣＲ－ＳＳＣＰ结果表明球形ＨＰ的ｈｐａＡ,ＶａｃＡ,ＣａｇＡ和ＵｒｅＡ４个毒力基因片段未发生缺失,但在ｈｐａＡ或ＶａｃＡ基因中存在点突变     

三种钠尿肽抑制大鼠肺动脉平滑肌细胞增殖效应的比较

本文比较了心房钠尿肽（ＡＮＰ）、Ｃ－型钠尿肽（ＣＮＰ）、血管钠肽（ＶＮＰ）抑制肺动脉平滑肌细胞（ＰＡＳＭＣｓ）增殖的效应。用蛋白激酶Ｃ激动剂佛波酯（ＰＭＡ）刺激体外培养大鼠ＰＡＳＭＣｓ的增殖,以总蛋白含量和ＭＴＴ比色ＯＤ值为指标,观察三种钠尿肽对ＰＭＡ刺激大鼠ＰＡＳＭＣｓ增殖的影响。结果表明,ＰＭＡ（１０＾－９－１０＾－７ｍｏｌ／Ｌ）显著升高（Ｐ＜０．０５）ＰＡＳＭＣｓ的总蛋白含量和ＭＴＴＯＤ值,     

蓝藻(Cyanobacteria)藻胆体内核结构与功能的研究I.变藻蓝蛋白六聚体(αβ)5APβ16.3LCM42分离及其表征

利用藻胆体温和解离的方法和超速离心分离技术首次得到了变藻蓝蛋白六聚体,并通过光谱技术,凝胶柱过滤柱色谱方法,和ＳＤＳ－ＰＡＧＥ电泳方法以及蔗糖密度超速离心方法对其进行表征。结果表明：该变落蓝蛋白六聚体的最大吸收峰（λ＾Ａｍａｘ＝６５２ｎｍ）,荧光发射峰位于６６６ｎｍ,并且在６８０ｎｍ处有一明显肩峰;其分子量大约为２４０ＫＤ左右;其分子组成为（αβ）５＾ＡＰβ＾１６．３ＬＣＭ＾４２;离心沉降系数为１     

大肠杆菌表达的重组细胞色素P—450nor的分离纯化

用ＤＥ－５２纤维素柱色谱法和ＦＰＬＣ法分离纯化了大肠杆菌表达的重组缣孢菌色素Ｐ－４５０ｎｏｒ，经梯度洗脱ＭｏｎｏＱ纯化后的ｆＲ．Ｐ－４５０ｎｏｒ为单一色谱峰，比活达５５．２０Ｕ／ｍｇ、纯化倍数约为１１００倍，ＳＤＳ－ＰＡＧＥ检测为单一谱带。     

丙型肝炎病毒基因组部份NS5区蛋白基因在大肠杆菌中的表达

在大肠杆菌中表达了丙型肝炎病毒基因组ＮＳ５区Ａ段和部份Ｂ段蛋白。ＮＳ５Ａ蛋白溶于水,可经过ＧＳＴ亲和层析柱纯化;ＮＳ５Ｂ蛋白不溶于水,经ＰＢＳ－Ｔｒｉｔｏｎ Ｘ－１００洗涤,尿素溶解后,通过离子交换柱纯化。经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｍｂｌｏｔ分析,ＮＳ５Ａ蛋白除在５７ｋＤ左右有条带外,还有不同程度的降解产物;ＮＳ５Ｂ蛋白主要在５８ｋＤ左右有条带出现。为查明表达蛋白抗体在病人血清中的分布,取９     

Crosslinking of concanavalin A with glutaraldehyde

Crosslinking of Concanavalin A with low concentrations of glutaraldehyde gives a mixture of products. A specific product having about 66% of the biological activity of the native molecule was characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed the presence of monomers, dimers, trimers, tetramers, and a small amount of pentamers as products. The presence of alpha-methyl mannoside during crosslinking changed the nature of the products, yielding a product retaining 80% of the biological activity. The crosslinked products showed greater stability than the native molecule at alkaline pH. However, the greatest stability under alkaline conditions was shown by the native molecule itself where alpha-methyl mannoside was present.     

人白血病细胞癌性促凝物(CP)的分离纯化及其特性的初步研究

用三步纯化法从人M_3型白血病细胞中分离纯化出人类肿瘤癌性促凝物(CP)。促凝活性回收率为24％,CP纯化倍数为2481倍。纯化CP在SDS-PAGE上为单一区带,其理化和酶学特性类似于动物肿瘤CP,分子量约为70 000,PI为4.8,在FVⅡ缺乏血浆中以及在含有组织因子(TF)抑制剂情况下仍能激活FX。CP促凝活性能被半胱氨酸蛋白酶抑制剂HgCl_2抑制,纯化CP能与抗动物肿瘤CP抗体形成免疫沉淀反应。     

以融合蛋白形式在大肠杆菌中表达人MCP－1

将编码人单核细胞趋化蛋白－１（ＭＣＰ－１）的基因亚克隆到大肠杆菌表达载体ｐＥＸ３１Ａ中,在大肠杆菌中表达出ＭＳ２／ＭＣＰ－１融合蛋０白,该表达产物约占菌体总蛋白的１５％左右,Ｗｅｓｔｅｒｎｂｌｏｔ检测表明,表达产物可与ＭＣＰ－１抗体特异反应。采用琼脂糖平板法进行活性测定表明,表达产物具有明显的单核细胞趋化活性,说明Ｎ端融合一段细菌蛋白对ＭＣＰ－１有无趋化活性可能没有影响。     

3α-羟类固醇脱氢酶基因的质粒载体构建和表达

目的 实现3α-羟类固醇脱氢酶基因在大肠埃希菌中的高可溶性表达.方法 从土壤中分离睾丸酮丛毛单胞菌,提取其基因组DNA,PCR扩增3α-羟类固醇脱氢酶(3α-HSD)基因,将它克隆到原核表达载体上进行诱导表达.提取细菌总蛋白进行SDS-PAGE分析并测定酶活性.结果 经核苷酸序列测定和酶切鉴定结果表明,成功地构建了重组质粒,IPTG诱导表达后,获得融合蛋白,SDS-PAGE初步测定目的蛋白的相对分子量约为29kDa,与预期理论值一致;酶活性测定结果表明菌体可溶性总蛋白HSD酶比活性为142.81 U/mg,是对照BL21的12.97倍.结论 该研究成功地构建了3α-羟类固醇脱氢酶基因高效原核表达系统,为利用基因工程手段大量制备3α-HSD的工作奠定了基础.     

Purification of isopenicillin N synthetase.

Isopenicillin N synthetase was extracted from Cephalosporium acremonium and purified about 200-fold. The product showed one major protein band, coinciding with synthetase activity, when subjected to electrophoresis in polyacrylamide gel. An isopenicillin N synthetase from Penicillium chrysogenum was purified about 70-fold by similar procedures. The two enzymes resemble each other closely in their Mr, in their mobility on electrophoresis in polyacrylamide gel and in their requirement for Fe2+ and ascorbate for maximum activity. Preliminary experiments have shown that a similar isopenicillin N synthetase can be extracted from Streptomyces clavuligerus.     

Genome-length copies of poliovirion RNA are synthesized in vitro by the poliovirus RNA-dependent RNA polymerase

A soluble RNA-dependent RNA polymerase was purified from the cytoplasm of poliovirus-infected HeLa cells. A single virus-specific protein designated as p63 (or NCVP4) copurified with this activity. The purified polymerase was free of ribonuclease activity and was shown to copy poliovirion RNA when oligo(U) was added to the in vitro reaction mixture. Characterization of the product RNA by electrophoresis in methylmercury (II) hydroxide-agarose gels showed that genome-sized copies of poliovirion RNA were synthesized in vitro by the purified polymerase. The product RNA was shown to be heteropolymeric, complementary to virion RNA, and covalently linked to oligo(U). The product RNA contained the expected distribution of UMP and GMP containing dinucleotide pairs which included a very low frequency of CpG pairs. The amount, size distribution, and rate of synthesis of product RNA was very dependent on the in vitro reaction conditions. Full sized product RNA was synthesized in about 6 min when reaction conditions were used that yielded maximum elongation rates (pH 8.0, 7 mM Mg2+, 37 degrees C). Under these conditions, most of the product RNA recovered from a 1-h reaction was full sized. Thus, the polymerase was found to specifically initiate synthesis at the 3'-end of the template using an oligo(U) primer and to carry out an elongation reaction at about 1250 nucleotides/min that resulted in the synthesis of full sized product RNA.     

蜂毒素分子的改造及其基因在毕赤酵母中的表达

为获得保留有抗菌活性而降低溶血作用的蜂毒素,对蜂毒素的分子结构进行了改造。将第5位的Val变为Arg,第15位Ala变为Arg,删除了第16位的Leu。用PCR技术获得了改造后的蜂毒素基因,将其克隆入酵母表达载体pPICZa-A,获得重组表达质粒pPICZa-A-MEA。该质粒转化毕赤酵母菌GS115,甲醇诱导下表达,发酵上清液经抑菌活性、溶血活性测定及亲和层析纯化,结果表明,蜂毒素基因成功地在毕赤酵母中表达,经改造后表达的蜂毒素保留了抗菌活性且溶血活性显著降低,经纯化后用Bradford法测定表达蜂毒素的含量约为0.29mg／ml。     

Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro.

A soluble RNA-dependent RNA polymerase was isolated from poliovirus-infected HeLa cells and was shown to copy poliovirus RNA in vitro. The enzyme was purified from a 200,000-X-g supernatant of a cytoplasmic extract of infected cells. The activity of the enzyme was measured throughout the purification by using a polyadenylic acid template and oligouridylic acid primer. The enzyme was partially purified by ammonium sulfate precipitation, glycerol gradient centrifugation, and phosphocellulose chromatography. The polymerase precipitated in a 35% saturated solution of ammonium sulfate, sedimented at about 7S on a glycerol gradient, and eluted from phosphocellulose with 0.15 M KC1. The polymerase was purified about 40-fold and was shown to be totally dependent on exogenous RNA for activity and relatively free of contaminating nuclease. The partially purified polymerase was able to use purified polio virion RNA as well as a template. Under the reaction conditions used, the polymerase required an oligouridylic acid primer and all four ribonucleside triphosphates for activity. The optimum ratio of oligouridylic acid molecules to poliovirus RNA molecules for priming activity was about 16:1. A nearest-neighbor analysis of the in vitro RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA rendered it resistant to RNase digestion, thus suggesting that the product RNA was complementary to the virion RNA template.     

Purification and some properties of cellulose 1,4-β-cellobiosidase from a strain of Penicillium sp.

A cellulase was purified from the culture supernatant of a strain of Penicillium sp. The purified enzyme was homogenous on polyacrylamide disc gel electrophoresis. It was a glycoprotein with a molecular weight of 52,000 estimated by gel filtration. The optimum pH was about 4.0 and the optimum temperature was 60°C. The enzyme was stable in the pH range of 3.0–10.0 at 6°C for 48 h and on heating at 60°C for 10 min. The activity of the enzyme toward Avicel was about 3 times higher than toward carboxymethyl cellulose. The enzyme showed a low activity for cotton, newspaper, filter paper and cellulose powder. The main product from Avicel was cellobiose, with a trace of glucose.     

猪α-干扰素的原核表达及活性测定

目的：表达并纯化出具有生物学活性的重组猪α-干扰素。方法：根据前期合成的猪α-干扰素基因序列设计引物,用PCR方法扩增出猪α-干扰素基因,并将其定向克隆入原核表达载体pET28中,酶切及测序鉴定正确后,转化大肠杆菌BL21进行诱导表达,对表达产物通过镍琼脂糖凝胶柱亲和层析纯化、透析法复性后,采用微量细胞病变抑制法测定重组猪α-干扰素的活性。结果：测序结果表明构建了猪α-干扰素原核表达载体pET28-poIFN-α;诱导表达后经SDS-PAGE分析,在相对分子质量约21×10^3的位置出现明显的诱导蛋白条带,与目的蛋白大小相近;经分析表达产物主要以包涵体形式存在,约占菌体总蛋白的36%,纯化后的目的蛋白纯度约为90%;采用微量细胞病变抑制法测定其活性约为1.67×10^6U/mg。结论：获得了纯度较高并具有较高活性的重组猪α-干扰素,为下一步研究猪α-干扰素药物价值及其生物制剂的生产、应用奠定了基础。