Objective: To study the agreement between chemiluminescence assay(Che) and bioluminescence imaging(Bio) used for detecting the firefly luciferase(Fluc) activity.Methods: The methods of chemiluminescence assay and bioluminescence imaging were used to detect the Fluc expression level at series time points after transfected with the vector pCI-AAA-Fluc-neo in which Fluc was as the report gene.Results: The results indicated that the Fluc expression level was decreasing as time going on.In HepG2 cell,the expression of Fluc lasted 96 h,the activity of Fluc decreased from 2781±220 mV(1.6×106±2.3×105 photons) at 24 h to 49±3.5 mV(6.4×104±2.5×104 photons) at 96 h.Similarly in vivo,the expression of Fluc of mice lasted 20 d,the activity of Fluc decreased from 16592±409 mV(1.9×108±3.6×106 photons) at 1 d to 798±139 mV(3.37×105±3.8×104 photons) at 20 d.Through the agreement study,the line regression equations between the two methods at different levels were gotten lgChe=1.186·lgBio-3.764(r=0.937,P<0.001,cell level) and lgChe=0.451·lgBio+0.64(r=0.915,P<0.001,animal level).Further t-test showed no significant between theory data(gotten from the line regression equations) and experimental data(P>0.05).Conclusion: The two methods are concordance with each other.Comparing the two methods,bioluminescence imaging is more convenient and direct.It also can detect the same individual continuously so as to greatly decrease the difference between individuals and usage amounts of animals.

Objective:To investigate the bioluminescent signals of orthotopic liver tumors in nude mice for noninvasive monitoring on progression of liver tumor in vivo. Methods:Human hepatocellular carcinoma cell line HepG2 cells were transduced with vector pCI-neo-Luc encoding luciferase gene. The expression of luciferase in HepG2 cells was analyzed with FACS and bioluminescence imagining. An orthotopic liver tumor model was created with cells from the heterotopic tumor established by implanting constitutively tumor cells expressing luciferase subcutaneously into nude mice. The tumor in vivo was monitored by whole-body images and quantity of photon emission. Results:In vitro result shows that photon emission correlated with increasing cell numbers. In vivo result indicates the establishment of an orthotopic liver tumor model expressing luciferase. Conclusion:This study provides a platform for monitoring the tumor by orthotopic implantation and evaluating in vivo efficacy of anti-tumor drugs.

This study was purposed to verify the binding part of human complement C3 to complement receptor III (CRIII) in monocytes, the peptide rC3B, including the binding-site, was expressed, purified and identified. rC3B, the binding part of human complement C3 to CRIII, was selected by computer-aided modeling and summarizing researches published. Then, rC3B gene fragment was amplified by PCR, and cloned into prokaryotic vector pQE30a. The fusion protein rC3B was expressed in E.coli M15 and purified by Ni(2+)-chelating affinity chromatography. The activity of rC3B was identified by Western blot and adherence assay with monocytes. The results showed that rC3B fragment was obtained, and a prokaryotic expression vector pQE30-rC3B was constructed. rC3B was efficiently expressed and purified. In Western blot, the target protein showed the activity of binding with C3 antibody, while the purified protein showed the activity of adherence with monocytes. It is concluded that the recombinant C3B was obtained and identified, and this study lay the basis for the further functional analysis of C3.

Objective: To isolate ribosome inactivating protein luffin-a gene from the seed of Luffa cylindrica and express it in E.coli.Methods:Luffin-a gene was obtained by RT-PCR using the primers designed according to known luffin-a(cDNA) sequence and cloned in pGEM-T vector.The luffin-a gene obtained was sequenced and analyzed.Expression vector pET-28a(+)-luffin-a was constructed and expressed in E.coli BL21(DE3).The interest protein was purified using immobilized metal ion affinity chromatography(IMAC).Results:The cDNA and amino acid sequence of the novel luffin-a obtained showed 95% and 92% homology with those reported by others.SDS-PAGE showed that the protein luffin-a was located in the inclusion body.The luffin-a protein could be purified by IMAC.Conclusion: A novel luffin-a gene is isolated,expressed in E.coli and purified by one-step IMAC.

Objective:To construct a liver highly-effective control element that can prolong the expression time of hydro-dynamically transfected gene.Methods:Plasmid pCIneo-AerApoEAATP-hAAT,pCIneo-ApoEAATP-hAAT,pCIneo-Al-bEAATP-hAATand pCIneo hAAT were constructed respectively.All of them included hAAT gene as the reporter gene.BALB/c mice were transfected with the plasmids by hydrodynamical transfection technology.The activity of AerApoEAATP,ApoEAATP,AlbEAATP and CMV was compared by the concentration of hAAT.Results and Conclusion:The control ele-ment constructed is much stronger than ApoEAATP,AlbEAATP and CMV in the long-term expression.

丙型病毒性肝炎是一种严重危害人类健康的常见病和多发病.全球约有1.7亿人感染丙型肝炎病毒(Hepatitis C Virus, HCV).我国HCV感染者已达6000万.长期以来,丙型肝炎的研究一直面临一大难题:即缺少稳定可靠的HCV感染细胞模型和动物模型,阻碍了对HCV致病机制、抗病毒药物筛选和疫苗研究等的研究进程.因此探索具有实际应用价值的HCV感染模型或HCV基因表达模型,是当今丙型肝炎防治研究的重大课题[1],近10年来众多研究者对此方面进行研究,本文即对此做一综述.

酶的体外定向进化是目前酶学工程发展的一个新技术,本文综述了易错PCR、DNA重组等策略的进展,该项技术为医学、工业、农业等方面提供了广阔的前景.

Objective: To verify the expression of the recombinant CB gene in mammalian cells,CB-GFP(green fluorescent protein)fusion gene eukaryotic expression vector pEGFP-N1-CB was constructed,and transfected into COS-7 cells.The expression of foreign gene was detected by laser confocus microscope.Methods:The encoding region of CB without terminator was obtained by PCR,and cloned into pGEM-T vector.After the double enzyme cutting,the recombinant CB gene was inserted into the expression vector pEGFP-N1.Thus,the plasmid pEGFP-N1-CB with fusion gene CB-GFP was constructed,and then transfected into COS-7 cells by liposome.The mRNA expression of CB was detected by RT-PCR.The expression and subcellular localization of the fusion protein was detected by Western blot and laser confocus microimaging.Results: RT-PCR verified CB mRNA expression in COS-7 cells.The efficient expression of CB-GFP fusion protein was observed and the fluorescence was mainly located in the cytoplasm of COS-7 cells.Conclusion: The recombinant CB protein can be expressed efficiently in mammalian cells,and this laid a foundation for the further functional researches on recombinant CB protein.

红细胞是一种天然免疫刺激剂和免疫调节剂,数量巨大的血红细胞在机体天然免疫中占有极其重要的地位.本文对红细胞免疫的研究历史、目前研究现状进行了较系统的阐述,并概括了近年来红细胞免疫研究的几大热点方向.

To express and purify the specific glycoprotein antigens of herpes simplex virus (HSV) as used in HSV clinical detection. HSV type-common glycoprotein gD gene and type-specific glycoproteins gG1(typeⅠ) and gG2 (typeⅡ) genes amplified by PCR were subcloned into prokaryotic vector pQE30 and expressed in E.coli M15. HSV glycoprotein gD was highly expressed and purified from the inclusion body. HSV specific glycoproteins were expressed successfully in prokaryotic system therefore provides the basis for further research on HSV clinical detection.

人类补体C3成分是补体激活的经典途径、替代途径以及甘露聚糖结合凝集素(MBL)途径三者的交汇点,是宿主防御机制中占据核心地位的特殊分子.补体C3与其裂解片段构成了人类补体系统的基础框架.现就近年来补体C3基因、蛋白、结合位点以及功能和应用相关的研究进展作一综述.

The aim was to develop a single multiple-epitope fusion antigen which incorporates all of the major immunodominant epitopes from the six functional regions of the HCV genome. A nucleic acid sequence consisting of viral core, E1, E2, NS3, NS4, and NS5 regions was constructed and inserted into the Promega Pinpoint Xa-1 T vector for inducing expression. The protein was expressed in JM109 (DE3) as a fusion protein with a 13 kD biotinlated tag to be used for detection and affinity purification. Immunogenicity and biotinylated tag of the fusion protein were detected by Western blot analysis with positive anti-HCV serum and streptavidin alkaline phosphatase. After purified by Promega SoftLink Soft Release Avidin Resin, the protein was pre-coated on microwell and detected with anti-core, anti-NS3, anti-NS4 and anti-NS5 positive sera by EIA, respectively. The results indicated that the recombinant soluble protein was expressed and labelled with biotin successfully, it reacted with anti-HCV positive serum, and exposed all of the major immunogenic epitopes chosen. In conclusion, this recombinant antigen may be used to design an double antigen sandwich anti-HCV immunoassay.

Virus inactivation of plasma can be achieved by methylene blue/photochemical method. To investigate the effect of this method on immunological properties and biochemical functions of plasma components, the virus-inactivation method was performed on single-donor plasma that was exposed to visible light (40,000 lux) at room temperature for 1 h in the presence of 1 micro mol/L methylene blue. The results showed that activities of the factor VIII, PT and APTT were decreased to a certain degree while most of other plasma proteins were not affected significantly. Human plasma components including albumin, glucose and minerals as well as plasma pH were also not affected. By using different electrophoreses and immunochemical techniques, no neoantigens were found in photodynamically treated plasma and electrophoretic mobility revealed identical patterns for untreated and treated plasma. In conclusion, methylene blue/photochemical method dose not considerably influence the properties of major of plasma components.

RT-PCR是检测病毒灭活过程中病毒RNA核酸链损伤的一种方法.常规的RT-PCR方法只能检测到病毒核酸受损伤的趋势,但不能精确地对损伤程度进行定量分析[1].笔者以竞争性RT-PCR方法, 通过构建一条与待扩增的目的基因具有相同扩增效率的竞争子片段,实现了对水泡性口膜炎病毒(vesicular stomatisis virus,VSV)核酸的初步定量,现将竞争子的制备方法介绍如下. 1 材料与方法

目的:了解光化学灭活病毒时,VSV核酸的动态变化.方法:利用来源于VSV核衣壳蛋白基因的一对引物以RT-PCR法检测VSV的核酸.结果:RT-PCR最大检测灵敏度为1.8Lg TCID50;在VSV光化学灭活过程中,RT-PCR产物量在5个灭活时间段上是不同的,随灭活时间的延长而减少.结论:光化学法对VSV核酸损伤程度随病毒灭活时间的延长而增大.