人免疫缺陷病毒Ⅰ型p24gag基因的重组与表达

半编码人Ⅰ型免疫缺陷病毒衣壳蛋白Ｐ２４＾ｇａｇ基因片段，克隆到原核表达载体ＰＥＴ－１７ｂ的Ｔ７噬菌体启动子下游。     

人骨形态发生蛋白－1（hBMP－1）成熟肽的基因克隆及其在大肠杆菌中的表达

采用ＰＣＲ方法从ＢＭＰ－１的ｃＤＮＡ中克隆了ＢＭＰ－１成熟肽的编码基因，删除了ＢＭＰ－１前体分子Ｎ端的信号肽序列和Ｃ端的其他序列。用ＨｉｎｄⅢ消化１．３Ｋｂ的ＰＣＲ产物，将０．８４和０．４６Ｋｂ两片段分别克隆到ｐＵＣ载体中进ＤＮＡ序列分析。分别酶切回收ＥｃｏＲ－ＨｉｎｄⅢ和ＨｉｎｄⅢ－ＢａｍＨⅠ两目的基因片段，与大肠杆菌表达载体ｐＢＶ－２２０进行退火连接，使插入基因受控于Ｐ＿ＲＰ＿Ｌ启动子。将含有ＢＭＰ－１成熟肽编码基因重组表达质粒ｐＢＶＢＭＰ－１转化大肠杆菌宿主细胞进行温度诱导表达。结果表明，经４２℃热诱导后，大肠杆菌能以包涵体形式表达ＢＭＰ－１成熟肽，其分子量约为５２ｋＤａ。     

CSF－1R部分基因在大肠杆菌中表达、抗融合蛋白血清制备及丝氨酸／苏氨酸磷酸化的初步研究

鼠巨噬细胞集落刺激因子－１受体（ｍＣＳＦ－１Ｒ）部分序列与质粒ｐＧＥＸ－２Ｔ谷胱苷肽转移酶（ＧＳＴ）融合，融合蛋白ＧＳＴ－ＣＤ－Ｐｓｔ（胞浆区），ＧＳＴ－ＣＴｅｒｍ（Ｃ－末端）和ＧＳＴ－ＫＩ（激酶插入区）成功地在大肠杆菌ＪＭ１０９株表达。初步结果指出：（１）ＧＳＴ－融合蛋白在体外激酶分析中可以作为底物；（２）由ＰＫＡ导致的磷酸化可能具有生理学意义；（３）ｍＣＳＦ－１Ｒ被ＣＫＩＩ磷酸化。３２Ｐ标记ＧＳＴ－ＣＤ－Ｐｓｔ的磷酸氨基酸分析证实，ｍＣＳＦ－１Ｒ的胞浆区丝氨酸上被磷酸化，已制备抗ＧＳＴ－ＣＴｅｒｍ，ＧＳＴ－ＫＩ和ＧＳＴ－ＣＤ－Ｐｓｔ兔抗体。抗血清的筛选通过野生型３２Ｄ－ＣＳＦ－１Ｒ转染子免疫沉淀进行。     

高表达重组人粒细胞集落刺激因子cDNA的中国仓鼠卵巢细胞株的建立

用质粒ｐＥＦ－ＢＯＳ的增强子、肽链延长因子基因的启动子及其第一内含子替换质粒ｐＥＤ－ＧＣＳＦ的增强子，腺病毒主要晚期启动子及杂合内含子。构建成了更高表达质粒ｐＥＦ－ＧＣＳＦ，转染ＣＨＯ－ｄｈｆｒ－细胞，加入并逐渐升高氨甲喋呤的浓度。结果显示：一定范围内，随着ＭＴＸ浓度增高，重组人粒细胞集落刺激因子表达量随之提高；筛选并建立了一株稳定高表达ｒｈＧ－ＣＳＦ ｃＤＮＡ的细胞株，其表达量为５．０￣５．６μ     

RP2突变体RP2-dser6的克隆和表达

视网膜色素变性是遗传性视觉损害以致失明的最常见原因之一,ＲＰ２是最近定位克隆的一个视网膜色素变性基因。为研究突变ＲＰ２蛋白与正常ＲＰ２蛋白的空间结构的差异及其对功能的影响,用突变的寡聚核苷酸引物自重组质粒ｐＥＴＲＰ２ ＰＣＲ扩增得到突变的ＲＰ２－ｄｓｅｒ６基因,克隆至ｐＥＴ１１Ｃ载体中,转化大肠杆菌ＢＬ２１菌株,重组子经序列分析鉴定后,在３７℃诱导Ｒ２－ｄｓｅｒ６突变蛋白表达,５ｍｍｏｌ／Ｌ ＩＰ     

恶性疟原虫FCC1／HN株P190抗原保守区基因的克隆及其在大肠杆菌中的表达

Ｐ１９０是恶性疟原虫裂殖子表面抗原，是很有希望的疟疾疫苗候选抗原。采用ＰＣＲ方法克隆了我国海南省ＦＣＣ１／ＨＮ株Ｐ１９０抗原基因的３个保守区片段，连接到ｐＵＣ１８载体上进行ＤＮＡ序列测定，然后分别与表达载体ｐＧＥＸ－２Ｔ连接，经双酶切鉴定后转化感受态ＪＭ１０９大肠杆菌进行高效融合表达。     

氯化聚乙烯及其接枝氯乙烯悬浮液粘度的研究

本文采用搅拌法测量氯化聚乙烯（ＣＰＥ）及氯化聚乙烯（ＣＰＥ）接枝（－ｇ－）氯乙烯（ＶＣ）悬浮液的粘度，与含固率建立关系，用Ｍｏｏｎｅｙ和Ｒｏｂｉｎｓｏｎ方程式进行关联。由悬浮液表观粘度ηａ与含固率ψｖ的关系曲线得出临界含固率ψｃ，结果发现ψｃ与悬浮液沉降层体积Ｖｐ呈线性关系：ＣＰＥ：ψｃ＝－０．４５５Ｖｐ＋１．４５６；ＣＰＥ－ｇ－ＶＣ：ψｃ＝－０．１８６Ｖｐ＋０．７５２     

运用一步化体外转录—翻译系统筛查基因突变的新技术

报道了一种筛查基因突变的新技术－ＰＣＲ结合一步化体外转转录－翻译系统。该技术包括：（１）ＰＣＲ扩增靶ＤＮＡ片段，并使ＤＮＡ产物的一端或两端带上Ｔ７Ｔ１序列；（Ｔ１：翻译起始信号，ＣＣＡ ＣＣＡ ＴＧ）；（２）以核的为模板，利用一步化体外转录－翻译系统合成相应肽链；（３）用ＳＤＳ－ＰＡＧＥ及高分辨ｐＨ梯度ＰＡＧＥ（聚丙烯酰胺凝胶电泳）分离分析多肽产物，通过异常电泳图谱而发现基因突变，本文以已知突变的     

表达人白细胞介素—2的重组腺病毒的制备

携带人白细胞介素－２ ｃＤＡ序列的Ｅ１区缺陷的腺病毒载体ｐＡｄｌ２／ＲＳＶ－ＩＬ２－ｂｐＡ与ｐＪＭ１７质粒，通过磷酸钙沉地共转染２９３细胞，经同源重组，获得了一个腺病毒噬斑。抽提腺病毒ＤＮＡ，运用ＰＣＲ扩增及ＸｂａＩ酶切检测，证实这一噬斑为带有人ＩＬ－２ ｃＤＮＡ序列的重组腺病毒。受其感染的２９３细胞和人黑素瘤Ａ３７５细胞上清中，都能够测出ＩＬ－２的活性，表明制备的重组腺病毒是能表达人ＩＬ－２的。     

CSF－1R激酶突变子的构建及其突变体在真核细胞的表达

通过ＰＣＲ二步法，构建了ｍＣＳＦ－１Ｒ激酶负性突变子，以及野生型和突变型ＣＳＦ－１Ｒ的送病毒表达载体ｐＣＥＮ／ＭＰＳＶ。进行了１２５Ｉ－ＣＳＦ－１受体结合分析：检测了激酶负性突变子，删除了ＣＳＦ－１ＲＣ－末端氨基酸９２５以后部分的突变体（ＣＴＲＵＮＣ９２５）和删除了激酶插入区的突变体（△ＫＩ）在３２Ｄ髓细胞表达。表达水平可达１～２×１０４受体／细胞。初步测定了通过ＣＳＦ－１Ｒ所介导的促细胞分裂效应。     

HIV-1核心蛋白的表达与纯化

将编码人I型免疫缺陷病毒(HIV1)核心蛋白p24gag的基因序列克隆到原核表达载体pET28(b)中,高效表达了N,C端融合His·Tagp24蛋白,所表达的重组p24蛋白占菌体总蛋白的46%。在变性条件下,使用NiNTA亲和层析法纯化了p24蛋白,纯度为94%。菌体中及纯化、复性后的目的蛋白均能与抗HIV1p24单克隆抗体发生特异性反应。用纯化的p24蛋白免疫小鼠,4周时小鼠血清抗p24抗体效价达1∶400。实验结果表明:大肠杆菌表达的HIV1p24蛋白纯化后可用作HIV1检测试剂的原料。     

Expression of the recombinant fusion protein CP15-23 of Cryptosporidium parvum and its protective test

The CP15 and CP23 surface proteins on the sporozoite of Cryptosporidium parvum are major protective antigens. The recombinant plasmid pET28-15-23 was constructed based on the plasmids pMD18-T-15 and pMD18-T-23 with two pairs of specific primers using DNA recombinant technique. In the primers, a synthetic linker sequence encoding a peptide (G-G-S) was designed. After identification, the recombinant plasmids were transformed to component cells of Escherichia coli BL21 (DE3). The positive strain containing the recombinant plasmid could express a specific fusion protein (CP15-23, MW approximately 25 kDa) induced by IPTG. The fusion protein could be recognized by the positive serum of mice infected with Cryptosporidium parvum oocysts specifically. The BALB/c mice were immunized with 80 microg of CP15-23 protein 4 times at 2 week intervals. The mice produced specific antibodies that responded to the lysate of Cryptosporidium parvum oocysts and could prevent Cryptosporidium parvum infection. The results indicated that the recombinant fusion protein CP15-23 would be used as a candidate antigen to prevent cryptosporidiosis.     

人体c-Mpl分子配体蛋白XP1的表达、纯化及活性初步鉴定

TPO是近年发现的血小板生成素，它通过与其受体c-Mpl的结合刺激巨核细胞的发生，调节动物或人体血小板的生成。利用酵母双杂交系统，以人体c-Mpl受体作为诱饵蛋白，我们从人体肝脏cDNA文库筛选到另一与c-Mpl相结合的新配体，命名为XP1。将：xpl-cDNA构建到pET-28b大肠杆菌融合表达载体，经金属离子亲和层析柱和DEAE阴离子层析柱分离纯化，获得纯度为99％的His-XPI水溶性融合蛋白。表达产物利用小鼠巨核细胞集落形成单位测定活性。结果表明：xpl基因在大肠杆菌中获得高效水溶性表达；其产物对小鼠骨髓细胞巨核细胞集落形成单位有明显的刺激作用。     

Controlled release of insulin from PLGA nanoparticles embedded within PVA hydrogels

A simple and versatile delivery platform for peptide and protein based on physically cross-linked poly (vinyl alcohol) (PVA) hydrogels containing insulin-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles was successfully fabricated. The particle morphology and size were characterized by SEM and laser light scattering method, respectively. Results showed that these particles had a mean diameter of 615 nm with a narrow size distribution and homogeneous particle production. The protein encapsulation efficiency was 72.6%. When insulin-loaded PLGA nanoparticles were administered intraperitoneally as a single dose (20 U/kg) to streptozotocin-induced diabetic mouse, blood glucose levels of these mice decreased and it could be sustained at such levels over 24 h. In vitro release further indicated that entrapment of the nanoparticles into the PVA hydrogels causes a reduction in both the release rate and the total amount of insulin released, which suggesting that PLGA nanoparticles entrapped into the PVA hydrogels showed more suitable controlled release kinetics for protein delivery.     

Ferricyanide reduction by Escherichia coli: kinetics, mechanism, and application to the optimization of recombinant fermentations

Ferricyanide reduction was studied by flow injection analysis (FIA) and chronoamperometry (CA) using two host strains and one recombinant strain of E. coli. Samples taken from batch cultures of E. coli JM105 and HB101 showed maximal specific ferricyanide reduction rates in the late exponential phase of growth, with values (micromol/min x g) of 24 (FIA) and 17 (CA) for JM105, and 36 (FIA) for HB101, when shake-flask cultures were sampled, and 70 for HB101, when a chemostat was used to control pH and dissolved oxygen concentration throughout the cultivation. Remarkably higher ferricyanide reduction rates were obtained with HB101 cells cultivated continuously at very slow growth rate, when chilled, resuspended cell samples were incubated for 5 min in solutions containing 10 mM succinate or formate. These compounds are substrates for primary, membrane-bound dehydrogenases that transfer electrons via ubiquinone to the cytochrome oxidase complexes. Apparent Michaelis-Menten kinetics were observed with respect to ferricyanide concentration when 10 mM succinate was included in the assay buffer; apparent Km values of 10.1+/-0.6 mM and 14.4+/-1.2 mM ferricyanide were obtained for exponential- and stationary-phase E. coli JM105, respectively. Cyanide inhibition studies show that ferricyanide is reduced mainly by cytochrome o oxidase in exponentially growing cells. The large difference in ferricyanide reduction rates observed in the absence and presence of succinate and formate were used to signal stationary-phase entry 5 h after induction of recombinant human Cu/Zn superoxide dismutase expression in a batch fermentation of E. coli HMS174(DE3)(pET3ahSOD). This new method can be used as an adjunct to the quantitation of medium components for the optimization of recombinant fermentations.     

基于蚕丝蛋白的低温保护剂用于梅山猪耳成纤维细胞的低温保存研究

细胞低温保存为临床治疗和科学研究提供优质的细胞。体积分数为10%二甲基亚砜(DMSO)和体积分数为20%胎牛血清(FBS)是目前冻存细胞常用的保护剂。但DMSO对细胞具有毒性损伤,FBS存在携带病毒、感染疾病的风险。本文以梅山猪耳成纤维细胞作为模型细胞进行冻存实验,将蚕丝蛋白用于低温保护剂中,用不同质量浓度的丝胶蛋白和不同体积分数的丝素蛋白来分别替代FBS和DMSO,验证其在细胞低温保存中的有效性。解冻复苏后用台盼蓝染色法、MTT法、24 h贴壁率等检测细胞的存活率和生长活力,筛选出最佳的基于天然蚕丝蛋白的低温保护剂配方。结果表明:含质量浓度为1%丝胶蛋白和含体积分数为20%FBS的低温保护剂对猪耳细胞的冻存效果无显著差异,说明丝胶蛋白能有效替代FBS。将体积分数为10%DMSO浓度降至5%,添加体积分数为10%丝素蛋白后,细胞的存活率和贴壁率与对照组相比无明显差异,说明丝素蛋白能降低DMSO的浓度。将质量浓度为1%丝胶蛋白与体积分数为10%丝素蛋白联用后,能达到较好的冻存效果。     

A MutS-based protein chip for detection of DNA mutations

This paper describes a new protein chip method for detection of single-base mismatches and unpaired bases of DNA, using a genetic fusion molecular system Trx-His6-Linker peptide-Strep-tagII-Linker peptide-MutS (THLSLM). The THLSLM coding sequence was constructed by attaching Strep-tag II and mutS gene to pET32a (+) sequentially with insertion of a linker peptide coding sequence before and behind Strep-tagII gene, respectively. THLSLM was expressed in E. coli AD494 (DE3) and purified using Ni(2+)-chelation affinity resin. THLSLM retained both mismatch recognition activity and streptavidin binding affinity. THLSLM was then immobilized on the chip matrix coated with streptavidin through the Strep-tag II-streptavidin binding reaction. The resulting protein chip was used to detect the mismatched and unpaired mutations in the synthesized oligonucleotides, as well as a single-base mutation in rpoB gene from Mycobacterium tuberculosis, with high specificity. The method could potentially serve as a platform to develop the high-throughput technology for screening and analysis of genetic mutations.     

Design of a 24-hour controlled porosity osmotic pump system containing PVP: formulation variables

Purpose: The purpose of this study was to design a 24-hour controlled porosity osmotic pump system that utilizes polyvinyl pyrrolidone (PVP) as an osmotic-suspending/release retarding agent of drugs. Methods: Osmotic tablet cores containing various ratios of ketoprofen and PVP were prepared by wet granulation and initially spray coated with similar solution of cellulose acetate. A formulation containing ketoprofen and PVP at a ratio of 1:7 was selected for further studies. Results: The final formulation containing PVP K-30 in the tablet core augmented the release of ketoprofen (poorly water-soluble) up to 90 % over 24 hours much higher than either PVP K-25 or PVP K-90 and retarded the release of pseudoephedrine HCl (highly water-soluble) up to 18 hours. Conclusion: This study proposed the dual use of PVP in osmotic pump systems containing solids to modulate the release of either poorly or highly water-soluble drug.     

Adhesive Bonding to Dentin Improved by Polymerizable Cyclodextrin Derivatives

The objective of this work was to determine bonding characteristics of a hydrophilic monomer formulation containing polymerizable cyclodextrin derivatives. The hypothesis was that a formulation containing hydrophilic cross-linking diluent comonomers and cyclodextrins with functional groups attached by hydrolytically stable ether linkages could form strong adhesive bonds to dentin. The previously synthesized polymerizable cyclodextrin derivatives were formulated with sorbitol dimethacrylate, methacrylic acid and phenylbis(2,4,6-trimethylbenzoyl) phosphine oxide photoinitiator. The same formulation without the polymerizable cyclodextrin derivatives isolated the effects of the polymerizable cyclodextrin derivatives. A commercial self-etching bonding system was tested as a comparative control. Ground mid-coronal dentin was etched with 37 % phosphoric acid (H₃PO₄) for 15 s and rinsed with distilled water for 10 s. Formulations were applied to the moist dentin and light-cured 10 s. A packable composite was then applied through irises and light-cured 60 s. Teeth were stored in water for 24 h before bonds were tested in a shearing orientation. One-way ANOVA was performed on the data. The average values of shear bond strengths were defined as loads at fracture divided by the 4 mm diameter iris areas. The average value of shear bond strength for the formulation containing the polymerizable cyclodextrin derivatives was higher (p < 0.05), where p is a fraction of the probability distribution) than that of the same monomeric formulation except that the polymerizable cyclodextrin derivatives were not included. This was supporting evidence that the polymerizable cyclodextrin derivatives contributed to improved bonding. The average value of shear bond strength for the formulation containing the polymerizable cyclodextrin derivatives was also higher (p < 0.05) than that of the commercial self-etching bonding system. These preliminary results are in accordance with the hypothesis that formulations containing polymerizable cyclodextrin derivatives can form strong adhesive bonds to hydrated dentin surfaces. Further improvements in bonding to hydrated biological tissues by use of advanced formulations are anticipated.