Systemic delivery of recombinant proteins by genetically modified myoblasts

The ability to stably deliver recombinant proteins to the systemic circulation would facilitate the treatment of a variety of acquired and inherited diseases. To explore the feasibility of the use of genetically engineered myoblasts as a recombinant protein delivery system, stable transfectants of the murine C2C12 myoblast cell line were produced that synthesize and secrete high levels of human growth hormone (hGH) in vitro. Mice injected with hGH-transfected myoblasts had significant levels of hGH in both muscle and serum that were stable for at least 3 weeks after injection. Histological examination of muscles injected with beta-galactosidase-expressing C2C12 myoblasts demonstrated that many of the injected cells had fused to form multinucleated myotubes. Thus, genetically engineered myoblasts can be used for the stable delivery of recombinant proteins into the circulation.     

Eukaryotic Expression and Activity Analysis of Capsid Gene of Swine Vesicular Disease Virus HK/70

The capsid protein precursor （P1）, which plays a major role for the generation of polypeptides of swine vesicular disease virus （SVDV）, was cloned from SVDV HK/70 strain into the retroviral vector pBABE puro and expressed in the mammalian cell line PK15 through the retroviral expression system. The activity of recombinant protein to induce immune response was evaluated in guinea pigs. IFA and Western Blot were used to detect the recombinant protein expression. The results showed that the recombinant protein could be recognized by SVDV positive serum, and animal test showed SVDV-specific antibodies. All of those results indicate that a retroviral-based vaccine carrying the capsid protein precursor （P1） of SVD is able to be expressed in the eukaryotic cell and elicites strong SVDV-specific immune responses in guinea pigs.     

High-level recombinant gene expression in rabbit endothelial cells transduced by retroviral vectors

By virtue of its immediate contact with the circulating blood, the endothelium provides an attractive target for retroviral vector transduction for the purpose of gene therapy. To see whether efficient gene transfer and expression was feasible, rabbit aortic endothelial cells were infected with three Moloney murine leukemia virus-derived retroviral vectors. Two of these vectors carry genes encoding products that are not secreted: N2, containing only the selectable marker gene neoR, and SAX, containing both neoR gene and an SV40-promoted adenosine deaminase (ADA) gene. The third vector, G2N, contains a secretory rat growth hormone (rGH) gene and an SV40-promoted neoR gene. Infection with all three vectors resulted in expression of the respective genes. A high level of human ADA expression was observed in infected endothelial cell populations both before and after selection in G418. G2N-infected rabbit aortic endothelial cells that were grown on a synthetic vascular graft continued to secrete rGH into the culture medium. These studies suggest that endothelial cells may serve as vehicles for the introduction in vivo of functioning recombinant genes.     

Site-specific gene expression in vivo by direct gene transfer into the arterial wall

A recombinant beta-galactosidase gene has been expressed in a specific arterial segment in vivo by direct infection with a murine amphotropic retroviral vector or by DNA transfection with the use of liposomes. Several cell types in the vessel wall were transduced, including endothelial and vascular smooth muscle cells. After retroviral infection, a recombinant reporter gene was expressed for at least 5 months, and no helper virus was detected. Recombinant gene expression achieved by direct retroviral infection or liposome-mediated DNA transfection was limited to the site of infection and was absent from liver, lung, kidney, and spleen. These results demonstrate that site-specific gene expression can be achieved by direct gene transfer in vivo and could be applied to the treatment of such human diseases as atherosclerosis or cancer.     

逆转录病毒载体介导的猪瘟病毒E2基因在真核细胞中表达及动物免疫试验

利用DNA重组技术将猪瘟病毒(CSFV)石门株囊膜蛋白E2基因插入逆转录病毒载体pBABE-puro中构建成重组逆转录病毒载体pBABE-puro-E2,该重组逆转录病毒表达载体与pVSVg质粒经磷酸钙共转染法将其转入293GP细胞中包装逆转录病毒假病毒。用包装的假病毒感染SP2/0细胞,经嘌呤霉素筛选阳性细胞后进行流式细胞技术(FACS)分析,结果表明CSFVE2基因在SP2/0细胞膜上成功表达。将表达E2蛋白的SP2/0细胞腹腔免疫BALB/c小鼠,用流式细胞仪检测证明,成功诱导小鼠产生了抗E2蛋白的抗体。为下一步利用细胞免疫小鼠研制抗猪瘟病毒单克隆抗体打下基础。     

Recombinant gene expression in vivo within endothelial cells of the arterial wall

A technique for the transfer of endothelial cells and expression of recombinant genes in vivo could allow the introduction of proteins of therapeutic value in the management of cardiovascular diseases. Porcine endothelial cells expressing recombinant beta-galactosidase from a murine amphotropic retroviral vector were introduced with a catheter into denuded iliofemoral arteries of syngeneic animals. Arterial segments explanted 2 to 4 weeks later contained endothelial cells expressing beta-galactosidase, an indication that they were successfully implanted on the vessel wall.     

Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein

Ribonuclease H digests the RNA strand of duplex RNA.DNA hybrids into oligonucleotides. This activity is indispensable for retroviral infection and is involved in bacterial replication. The ribonuclease H from Escherichia coli is homologous with the retroviral proteins. The crystal structure of the E. coli enzyme reveals a distinctive alpha-beta tertiary fold. Analysis of the molecular model implicates a carboxyl triad in the catalytic mechanism and suggests a likely mode for the binding of RNA.DNA substrates. The structure was determined by the method of multiwavelength anomalous diffraction (MAD) with the use of synchrotron data from a crystal of the recombinant selenomethionyl protein.     

PEDV ps420片段乳酸乳球菌表达及免疫中和活性检测

将猪流行性腹泻病毒LJB/03株S基因上编码中ps420(1 495～1 916 bp)基因片段插入乳酸乳球菌pNZ8112中,构建了重组表达载体pNZ8112-ps420,将其电转化入乳酸乳球菌Lactococcus lactis NZ9000,获得了表达猪流行性腹泻病毒S基因ps420的重组乳酸乳球菌,经乳链菌肽(...     

Dimerization of human growth hormone by zinc

Size-exclusion chromatography and sedimentation equilbrium studies demonstrated that zinc ion (Zn2+) induced the dimerization of human growth hormone (hGH). Scatchard analysis of 65Zn2+ binding to hGH showed that two Zn2+ ions associate per dimer of hGH in a cooperative fashion. Cobalt (II) can substitute for Zn2+ in the hormone dimer and gives a visible spectrum characteristic of cobalt coordinated in a tetrahedral fashion by oxygen- and nitrogen-containing ligands. Replacement of potential Zn2+ ligands (His18, His21, and Glu174) in hGH with alanine weakened both Zn2+ binding and hGH dimer formation. The Zn(2+)-hGH dimer was more stable than monomeric hGH to denaturation in guanidine-HCl. Formation of a Zn(2+)-hGH dimeric complex may be important for storage of hGH in secretory granules.     

In Vivo genetic transformation of chiken spermatogonia

The efficiency of recombinant DNA transfer into chicken spermatogonia in vitro and in vivo with the use of retroviral vectors is studied.     

重组逆转录病毒载体系统的建立及其感染滴度的研究

选择新霉素磷酸转移酶II（Neo^R)做为标记基因,将重组逆转录病毒载体经包装细胞馐后得到的重组病毒浓缩后,应用该重组逆转录病毒载体系统体外感染靶细胞NIH3T3以测定其滴度,其滴度可达到10^6,具有理想的感染靶细胞的能力。     

丙型肝炎病毒囊膜蛋白基因E1E2在真核细胞中的表达及动物免疫试验

利用DNA重组技术将丙型肝炎病毒（HCV）H77株E1E2囊膜蛋白基因插入逆转录病毒载体pBABE-puro中，构建成重组逆转录病毒载体pBABE-puro-E1E2，用重组逆转录病毒载体与pVSVg质粒磷酸钙共沉淀法转染293T细胞,包装逆转录病毒假病毒。用包装的假病毒感染SP2/0细胞，经嘌呤霉素筛选阳性细胞后进行流式细胞仪技术（FACS）分析，结果表明,HCV E1E2基因在SP2/0细胞膜上成功表达。将表达E1E2蛋白的SP2/0细胞腹腔免疫BALB/c小鼠，经FACS分析免疫鼠血清，成功诱导小鼠产生了抗HCV E1E2蛋白的抗体，Western blot检测结果表明该抗体能与原核系统表达的E2蛋白结合。     

Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule

Human growth hormone (hGH) forms a 1:2 complex with the extracellular domain of its receptor-binding protein (hGHbp) as studied by crystallization, size exclusion chromatography, calorimetry, and a previously undescribed fluorescence quenching assay. These and other experiments with protein engineered variants of hGH have led to the identification of the binding determinants for two distinct but adjacent sites on hGH for the hGHbp, and the data indicated that there are two overlapping binding sites on the hGHbp for hGH. Furthermore, the binding of hGH to the hGHbp occurred sequentially; a first hGHbp molecule bound to site 1 on hGH and then a second hGHbp bound to site 2. Hormone-induced receptor dimerization is proposed to be relevant to the signal transduction mechanism for the hGH receptor and other related cytokine receptors.     

Implantation of genetically engineered fibroblasts into mice: implications for gene therapy

In a variety of human genetic diseases, replacement of the absent or defective protein provides significant therapeutic benefits. As a model for a somatic cell gene therapy system, cultured murine fibroblasts were transfected with a human growth hormone (hGH) fusion gene and cells from one of the resulting clonal lines were subsequently implanted into various locations in mice. Such implants synthesized and secreted hGH, which was detectable in the serum. The function of the implants depended on their location and size, and on the histocompatibility of the donor cells with their recipients. The expression of hGH could be modified by addition of regulatory effectors, and, with appropriate immunosuppression, the implants survived for more than 3 months. This approach to gene therapy, here termed "transkaryotic implantation," is potentially applicable to many genetic diseases in that the transfected cell line can be extensively characterized prior to implantation, several anatomical sites are suitable for implantation, and regulated expression of the gene of therapeutic interest can be obtained.     

Cloning and Expression of One Fibrinolytic Enzyme from Bacillus sp. zlw-2

The gene encoding fibrinolytic enzyme from Bacillus sp. zlw-2 was cloned and sequenced （accession no. EU734749）, which was 1146 bp, encoded 381 amino acids and had 99% homology with Nattokinase YF308 and NAT. The genes encoding pre-pro-fibrinolytic enzyme （including signal peptide, propeptide, and mature peptide） and fibrinolytic enzyme （including mature peptide） were cloned into pET28a vector respectively and then transformed into Escherichia coli BL21 （DE3）. The recombinant ofpre-pro-fibrinolytic enzyme showed enzyme activity of 183 U mL^-1, while no detectable enzyme activity could be found from the recombinant of the mature peptide.     

日本血吸虫Sj CyclophilinA基因的克隆、表达及其生物学功能研究

【目的】克隆和表达了日本血吸虫CyclophilinA(Sj CyPA)编码基因cDNA,分析其在日本血吸虫不同发育阶段虫体的表达情况,评估该重组抗原在小鼠体内诱导的抗血吸虫免疫保护效果。【方法】从实验室构建的7天童虫消减cDNA文库中,PCR扩增一EST序列的基因全长cDNA,提交到NCBI,登录号为GQ403666。应用荧光实时定量PCR分析该基因在日本血吸虫不同发育阶段虫体的表达情况,以pET28a(+)为载体构建重组表达质粒,并在大肠杆菌中表达。诱导、表达、纯化和复性重组蛋白,测定其PPIase活性。利用Western blot检测重组蛋白的抗原性。以重组抗原免疫小鼠,评估其对小鼠诱导的免疫保护效果。【结果】PCR获得了Sj CyPA编码基因的全长cDNA,其开放阅读框为519bp。荧光实时定量PCR分析表明,该基因在13d童虫表达量最高,为童虫期高表达基因。构建了重组表达质粒pET28a(+)-Sj CyPA,并在大肠杆菌中成功表达。复性重组蛋白具有PPIase活性。Western blot试验显示该重组蛋白具有良好的抗原性,在小鼠免疫试验中,与空白对照组比较,免疫组小鼠获得18.72%的减虫率和44.6%的肝脏减卵率。【结论】获得了日本血吸虫童虫期高表达的Sj CyPA基因的全长cDNA,成功构建了Sj CyPA原核重组表达质粒,在大肠杆菌中成功表达,纯化复性得到有PPIase活性的Sj CyPA重组蛋白,并证实该重组抗原在小鼠体内诱导产生了部分免疫保护效果。     

Designing small-molecule switches for protein-protein interactions

Mutations introduced into human growth hormone (hGH) (Thr175 --> Gly-hGH) and the extracellular domain of the hGH receptor (Trp104 --> Gly-hGHbp) created a cavity at the protein-protein interface that resulted in binding affinity being reduced by a factor of 10(6). A small library of indole analogs was screened for small molecules that bind the cavity created by the mutations and restore binding affinity. The ligand 5-chloro-2-trichloromethylimidazole was found to increase the affinity of the mutant hormone for its receptor more than 1000-fold. Cell proliferation and JAK2 phosphorylation assays showed that the mutant hGH activates growth hormone signaling in the presence of added ligand. This approach may allow other protein-protein and protein-nucleic acid interactions to be switched on or off by the addition or depletion of exogenous small molecules.     

牛fabp3和fabp4基因真核表达载体的构建 及在小鼠成肌细胞中的表达

【目的】分别构建牛fabp3和fabp4基因真核表达载体,并观察载体转染小鼠成肌细胞后基因的表达以及对内源fabp3和fabp4基因表达的影响,检测肉质性状形成相关基因在转基因细胞中的表达及对内源功能基因的影响。【方法】 以pDsRED质粒为骨架载体,分别将人肌红蛋白启动子与目的基因相连、CMV启动子与红色荧光蛋白报告基因相连构建肌肉特异性表达fabp3基因的载体pDsHF3和表达fabp4基因的载体pDsHF4;用脂质体瞬时转染小鼠成肌细胞,72 h后用2%孕马血清诱导成肌细胞向肌管的分化;利用real-time PCR技术检测转染前后成肌细胞中外源牛fabp3和fabp4基因和内源小鼠fabp3和fabp4基因的表达量。【结果】与对照组相比,小鼠成肌细胞分别转染pDsHF3和pDsHF4表达载体,24 h后外源牛fabp3和fabp4基因均获得高水平的表达,48 h后有所回落;而内源小鼠fabp3和fabp4基因在成肌细胞和诱导分化的肌管细胞中的表达均受到不同程度的影响。【结论】构建的真核表达载体能在成肌细胞中高效表达,外源牛fabp3或fabp4基因的转入能够影响小鼠成肌细胞及肌管中内源fabp3和fabp4基因的表达。     

Zinc mediation of the binding of human growth hormone to the human prolactin receptor

Human growth hormone (hGH) elicits a diverse set of biological activities including lactation that derives from binding to the prolactin (PRL) receptor. The binding affinity of hGH for the extracellular binding domain of the hPRL receptor (hPRLbp) was increased about 8000-fold by addition of 50 micromolar ZnCl2. Zinc was not required for binding of hGH to the hGH binding protein (hGHbp) or for binding of hPRL to the hPRLbp. Other divalent metal ions (Ca2+, Mg2+, Cu2+, Mn2+, and Co2+) at physiological concentrations did not support such strong binding. Scatchard analysis indicated a stoichiometry of one Zn2+ per hGH.hPRLbp complex. Mutational analysis showed that a cluster of three residues (His18, His21, and Glu174) in hGH and His188 from the hPRLbp (conserved in all PRL receptors but not GH receptors) are probable Zn2+ ligands. This polypeptide hormone.receptor "zinc sandwich" provides a molecular mechanism to explain why nonprimate GHs are not lactogenic and offers a molecular link between zinc deficiency and its association with altered functions of hGH.