免疫抑制蝎毒活性肽Im169的重组表达和功能鉴定

目的:筛选鉴定新型蝎毒免疫活性肽。方法:从海南斑等蝎(Isometrus maculates)毒腺组织c DNA文库中筛选和分离蝎毒肽基因,通过GST融合蛋白表达纯化、肠激酶酶切和高压液相色谱(HPLC)分离的方法,获得色谱纯重组蝎毒活性肽,酶联免疫吸附(ELISA)实验检测细胞因子,鉴定免疫调节活性。结果:筛选得到一个新的蝎毒活性肽基因Im169,其编码的成熟肽由29个氨基酸组成,其中含有6个半胱氨酸。Im169多肽对T淋巴细胞IL-2、TNF-α和IFN-γ等细胞因子的分泌具有明显的抑制作用,且作用呈浓度依赖性(P<0.05)。序列分析显示:Im169多肽和蝎钾通道毒素的相似性最高,提示其是一个潜在的钾通道毒素多肽。功能机制分析显示:Im169多肽通过抑制T细胞表面的钾通道,从而发挥免疫抑制功能。结论:发现了一个新的蝎毒免疫调节肽,丰富了对蝎毒活性肽的功能认识,为自身免疫疾病的药物开发提供了新的多肽模板。     

Anti-obesity effects of KR-66195, a synthetic DPP-IV inhibitor,in diet-induced obese mice and obese-diabetic ob/ob mice

Objective

We investigated whether KR-66195, a new synthetic dipeptidyl dipeptidase IV inhibitor, could prevent weight gain, as well as improving glycemic control in diet-induced obese (DIO) and ob/ob mice.

Materials/Methods

Male C57BL/6 mice were randomly assigned to the following groups: chow diet, high-fat diet, and high-fat diet with KR-66195. After KR-66195 treatment for eight weeks, intraperitoneal glucose tolerance tests were performed. A pair-feeding study was performed to investigate the mechanisms of the anti-obesity effects of KR-66195 in DIO mice. Female ob/ob mice were treated with KR-66195 for three weeks and compared to the vehicle-treated group.

Results

In DIO mice, KR-66195 treatment increased the plasma glucagon-like peptide (GLP)-1 levels and improved glucose tolerance. This treatment also reduced body weight gain (5.38 ± 0.94 g vs. 12.08 ± 0.55 g, P < 0.01) and food intake (2.41 ± 0.09 g vs. 2.79 ± 0.11 g, P < 0.05). In ob/ob mice, KR-66195 treatment for three weeks resulted in comparable effects in DIO mice. In the pair-feeding study, KR-66195-treated mice exhibited a 16% increase in energy expenditure (kcal/h/kg lean body mass) without significant differences in body weight or activities compared with pair-fed mice. These results suggest that KR-66195 prevented weight gain in DIO mice by decreasing food intake, as well as increasing energy expenditure.

Conclusions

KR-66195 markedly increased plasma levels of GLP-1, resulting in the probable improvement in glucose tolerance and reduced body weight and food intake. Thus, KR-66195 might be further developed as a therapeutic drug to treat obesity, as well as diabetes.     

Generation of a cholangiocyte-specific cDNA expression library for the identification of B and T cell autoantigens in murine biliary disease

Aim: Several mouse models of inflammatory cholangiopathies exist, including biliary atresia, primary biliary cirrhosis, autoimmune hepatitis, and primary sclerosing cholangitis. In an ongoing effort to identify the target antigens of both infiltrating autoreactive T cells and serum autoantibodies, we aimed to generate a cholangiocyte‐derived cDNA library capable of expressing a wide variety of proteins. Methods: mRNA was isolated from a normal mouse cholangiocyte cell line and reverse transcribed into cDNA. After initial cloning of the cDNA into a transfer vector (pDONR222), the entire library was shuttled into an Escherichia coli expression vector (pDEST160). Results: The library contains 2.3 × 10⁶ independent clones and expresses proteins up to 100 kD in molecular weight. Using a variety of techniques, including western blot analysis, mass spectrometry of individual clones, and direct DNA sequencing of plasmids, a number of both ubiquitously expressed and cholangiocyte‐specific proteins (e.g. cytokeratin 19) have been identified within. Conclusion: A comprehensive mouse cholangiocyte cDNA expression library has been generated and is available for use as a source of multiple cholangiocyte‐specific antigens for immunological studies. The library can be used to screen for specificity of T cell lines or hybridomas. Furthermore, this library has potential uses in SEREX analysis of autoantibody reactivity. The cholangiocyte‐specific cDNA library is a powerful tool for the identification of target antigens in murine inflammatory cholangiopathies and is available as a shared resource.     

Replication-competent recombinant porcine reproductive and respiratory syndrome (PRRS) viruses expressing indicator proteins and antiviral cytokines

Porcine reproductive and respiratory syndrome virus (PRRSV) can subvert early innate immunity, which leads to ineffective antimicrobial responses. Overcoming immune subversion is critical for developing vaccines and other measures to control this devastating swine virus. The overall goal of this work was to enhance innate and adaptive immunity following vaccination through the expression of interferon (IFN) genes by the PRRSV genome. We have constructed a series of recombinant PRRS viruses using an infectious PRRSV cDNA clone (pCMV-P129). Coding regions of exogenous genes, which included Renilla luciferase (Rluc), green and red fluorescent proteins (GFP and DsRed, respectively) and several interferons (IFNs), were constructed and expressed through a unique subgenomic mRNA placed between ORF1b and ORF2 of the PRRSV infectious clone. The constructs, which expressed Rluc, GFP, DsRed, efficiently produced progeny viruses and mimicked the parental virus in both MARC-145 cells and porcine macrophages. In contrast, replication of IFN-expressing viruses was attenuated, similar to the level of replication observed after the addition of exogenous IFN. Furthermore, the IFN expressing viruses inhibited the replication of a second PRRS virus co-transfected or co-infected. Inhibition by the different IFN subtypes corresponded to their anti-PRRSV activity, i.e., IFNω5 ° IFNα1 > IFN-β > IFNδ3. In summary, the indicator-expressing viruses provided an efficient means for real-time monitoring of viral replication thus allowing high‑throughput elucidation of the role of host factors in PRRSV infection. This was shown when they were used to clearly demonstrate the involvement of tumor susceptibility gene 101 (TSG101) in the early stage of PRRSV infection. In addition, replication‑competent IFN-expressing viruses may be good candidates for development of modified live virus (MLV) vaccines, which are capable of reversing subverted innate immune responses and may induce more effective adaptive immunity against PRRSV infection.     

牛带绦虫成虫钙调神经磷酸酶B基因生物信息学分析

目的 从牛带绦虫(Teania saginata)成虫cDNA文库中克隆牛带绦虫钙调神经磷酸酶B(calcineurin B钙调神经磷酸酶B)基因,分析和预测其编码蛋白的结构和特性.方法 构建牛带绦虫成虫cDNA质粒文库,从文库中识别钙调神经磷酸酶B基因并进行生物信息学分析.结果 该基因全长510 bp,编码区为141～650,编码170个氨基酸,为全长基因.理论分子量、等电点分别为19119.3 Da和4.52;无跨膜区;三维结构建模图显示其线性表位的部位.结论 应用生物信息方法从牛带绦虫成虫cDNA文库中筛选出钙调神经磷酸酶B基因的cDNA全长序列,并预测得到其结构与功能方面的信息.     

兄妹肾阳虚证温补作用的系统生物学研究

目的:引入系统生物学的观察方法,研究温补药物治疗肾阳虚证兄妹疗效的分子生物学特征,探索基因芯片在中医诊治学中应用的可能性。方法:用40项肾阳虚证量表和40项寒证量表,调查一对兄妹在治疗前后的症状积分;用18816点阵的基因芯片测试在治疗前后的基因表达水平。结果:评定兄妹温补之后肾阳虚与寒证症状的积分下降1/3～2/3,得到在兄妹治疗前的差异表达治疗后入正常值的84条共同基因,其中38条已知基因功能,包括免疫、生长发育等6类。讨论:肾阳虚与寒证治疗效果可以用38条共同调节的基因表达水平和量表积分来分析,两者之间的相关关系值得深入探讨。从系统生物学的观点,用基因差异表达来评价辨证治疗疗效,为中医证的疗效评价提供了一个新的方法。     

人胃癌细胞定向克隆表达cDNA文库的构建及鉴定

目的：克隆胃癌的相关基因。方法：提取人胃癌细胞总RNA，分离其mRN。以NatI／oligo（dT）12-18为引物合成双链。cDNA，除去小于400bp的cDNA片段后，连接EcoRⅠ人工接头，经NotⅠ酶酶切，插入定向克隆表达载体λExcellNotI／EcoRI／CIP，体外包装后转染大肠杆菌NM522，构建了人胃癌细胞MGC－803定向克隆表达。cDNA文库。结果：文库含1．2×106个重组子，重组率为96．7％。用PCR技术鉴定，文库中插入cDNA的平均大小为1kbc结论：该文库适于筛选各种丰度mRNA的cDNA克隆。