抗菌肽对病原菌的作用模式不同于传统抗生素,且能对耐药菌株产生抗性.抗菌肽不仅有广谱的抗菌活性,还能抗病毒、真菌和原虫,以及有强大的免疫调节活性.此外,抗菌肽还有促进创面愈合,加强皮肤屏障功能等作用.研究人员一直致力于发现各种内源性抗菌肽,并寻找将其规模化表达的方法.我们主要总结了内源性抗菌肽(主要是哺乳动物)的分类,作用机制及其免疫调节方面的重要作用.另外,介绍了目前提纯内源性抗菌肽,人工合成抗菌肽(如纳米抗菌肽)的研究成果和遇到的问题.

OBJECTIVE To construct a recombinant lentiviral vector expressing small-hairpin RNA (shRNA) targeting human Wnt5a gene and investigate its silencing effect on WM793B human melanoma invasion. METHODS Based on the sequence of human Wnt5a gene in GenBank, Wnt5a siRNA was designed and synthesized. The single-stranded primers were annealed to double-stranded oligonucleotide sequences and subcloned into linear pLKO.1 lentiviral plasmid digested by enzyme to produce pLKO.1-shWnt5a lentiviral vector. After being identified by PCR and sequencing, plasmid pLKO.1-shWnt5a was transfected into HEK293T cells to package lentiviral particles. Human malenoma WM793B cells were infected by the lentiviral particles. Expression of shWnt5a in WM793B cells was detected using Western blotting. Then the Transwell(TM); invasion assay was performed to assess its effect on melanoma cell invasion. RESULTS Lentivirus expressing shWnt5a was successfully constructed and WM793B(Wnt5a-);, a strain of melanoma cells with low expression of Wnt5a was also established. Transwell(TM); invasion assay revealed that cell migration was inhibited in Wnt5a-inhibited melanoma cells. CONCLUSION Down-regulated Wnt5a expression exerts a significant inhibitory effect on the invasion of melanoma cells.

AIM: To investigate whether bicistronic lentiviral vectors containing TFR and VEGF genes can mediate the expression of TFR and VEGF genes in endothelial progenitor cells(EPCs) of Chinese mini-swine.METHODS: TFR,IRES and VEGF genes were cloned into pLenti-GFP-Neo to generate the lentiviral vector pLenti-GFP-TIR.We sucessfully cultured EPCs of Chinese mini-swine where we identified the expression of cell surface CD31 and Flk-1 by flow cytometry(FCM).The four-plasmid lentiviral vector system(pRsv-REV,pMDlg-pRRE,pMD2G and pLenti-GFP-TIR) was cotransfected into human embryonic kidney 293T cells using the Lipofectin 2000 method.The packaged virus was harvested 72 h later and EPCs were infected by lentivirus carrying TFR and VEGF genes.RT-PCR was used to detect expression of TFR and VEGF genes in the viral-infected EPCs.RESULTS: EPCs were successfully infected by the recombinant lentivirus.TFR and VEGF genes were detected in the infected EPCs by RT-PCR.CONCLUSION: The bicistronic lentiviral vector containing TFR and VEGF genes can effectively deliver genes into EPCs and express target genes,providing the basis for future research on cardiac molecular imaging of cell transplantation.

Restin, a novel member of the melanoma-associated antigen super family, is cloned from differentiated leukemia cells induced by retinoic acid. Restin may interact with some transcription factors. The present study investigated the effect of restin on activating transcription factor 3 (ATF3), an important protein in numerous physiological situations, using the mammalian two-hybrid system. The results show that ATF3 strongly interacted with restin. To determine the effect of restin on ATF3 activity, an ATF3 reporter plasmid with a luciferase gene was constructed. Hela and NIH3T3 cells transfected with both restin and ATF3 exhibited accelerated ATF3 activity compared with those transfected with only ATF3. This result suggests the role of restin in facilitating ATF3 activity. Restin was also shown to up-regulate ATF3 activity by its interaction with the ATF3 DNA binding domain. Overall, these data suggests the involvement of restin in important cellular modulation processes in Hela and NIH3T3 cells by enhancing ATF3 activity. Key words: Restin, MAGE, ATF3, two-hybrid system, ATF3 reporter, luciferase.

AIM:To construct a dual promoter lentivira vector containing TFR and VEGF gene, and detect the expression of TFR and VEGF genes in MSCs of Chinese mini-swine.METHODS:The TFR and VEGF gene were amplified by polymerase chain reaction and cloned into pLenti-GFP-Neo after the CMV and SV40 promoter respectively, to generate the lentivira vector pLenti-TG-VEGF. The four-plasmids lentiviral vector system(pRsv-REV, pMDlg-pRRE, pMD2G and pLenti-TG-VEGF)were cotransfected into human embryonic kidney 293 T cells with Lipofectin 2000 reagent. The packaged virus was harvested 72 h later. MSCs were infected by lentivirus carrying TFR and VEGF genes. Western blotting was used to detect the expression of TFR and VEGF genes in the viral infected MSCs.RESULTS:Restriction endonuclease digestion analysis and DNA sequencing demonstrated that the dual promoter lentivirus vector containing TFR and VEGF genes were constructed successfully. MSCs could be successfully infected by the recombinant lentivirus. Expression of TFR and VEGF protein could be detected in the infected MSCs by Western blotting.CONCLUSION:The dual promoter lentiviral vector containing TFR and VEGF genes are constructed successfully which provides basis for future research on Cardiac molecular imaging of cell transplantation.

为适应新的教育改革需要和医学八年制学生培养目标定位高的特点,倡导生物化学与分子生物学实验技术课程针对学生采取不同的授课方式。系统化地集中教授理论部分,充分利用课堂时间,创造更多的实践机会,实现课堂效果最大化,集中时间,利用大型综合实验的开展,提高学生综合实践能力。

针对高校分子生物学实验室存在的污染问题,从废气、废水、固体废弃物及辐射这四个方面进行分析,阐明实验室污染种类和危害,提出了应从培养环保意识、健全规章制度、改进教学实验方法和完善实验室建设方面解决实验室的污染问题,并进行详细阐述。呼吁广大科研人员积极加入环保行列,为维护人类的居住环境贡献一份力量。

Objective:To construct the recombinant plasmid of human Restin and express the protein in a prokaryotic system. Methods:Human Restin gene coding region was obtained by PCR method and was cloned into pET44a(+)vector,a prokaryotic expression vector.The plasmid was transformed into E.coli BL21(DE3)and E.coli Rosetta-gamiTM2(DE3)to express Restin fusion protein in response to IPTG induction.The products were analyzed by SDS-PAGE and Western-Blot.Results:The recombinant plasmid pET44a(+)-Restin was constructed and the fusion protein was expressed in E.coli.And the molecular mass was about 97 kD and presence of soluble protein in E.coli Rosetta-gamiTM2(DE3).Conclusion:The soluble fusion protein of Restin in E.coli were expressed successfully, which establish a basis for further study about biological functions of Restin.

Aim: To prepare anti-human ameloblastin antibody, by which to study the tissue specificity of human ameloblastin. Methods: The experiment was performed in the Basic Department of Biochemistry and Molecular Biology of Fourth Military Medical University in March 2002. The C telopeptide of recombinant purified ameloblastin was used as the antigen to mixed with complete/incomplete Freund's adjuvant. The blood sample was obtained from the carotid artery (CA) of immune New Zealand white rabbits after 5 times of immunity, and the serum was separated purified with saturated ammonium sulphate to establish rabbit-anti-human ameloblastin multiclonal antibody. The titer of antibody was detected by double immunodissfusion test and ELISA. The tissue expression specificity of human ameloblastin antibody was determined by Western Blot. Results: 1 ELISA detection showed that the titer of the rabbit-anti-human ameloblastin multiclonal antibody was about 1:10 000. 2 Western Blot indicated that the ameloblastin was specifically expressed in the tooth germ with a relative molecular mass of 65 000, but no expression-strap was found in the brain, heart, liver, spleen, lung, kidney, pancreas, thymus or skeletal muscle of human. Conclusion: The anti-human ameloblastin antibody is successfully prepared, which provide a basis for the research on the tissue expression of ameloblastin in the tooth germ as well as the purification of protein by the antibody, and it is proved from the aspect of protein that the ameloblastin is the specific protein of tooth tissue, moreover, the relative molecular mass of ameloblastin in human tooth germ was about 65 000.

AIM: To investigate the effect of the cyclic tension force on the osteoclast formation in human bone marrow culture,so as to learn about the mechanisms of bone remodeling under mechanical strain.METHODS:Osteoclasts were cultured in marrow cultures plated on flexible membranes. Comparative proteomic approach based on two dimensional gel electrophoresis, Mass-spectrum identification and bioinformatics analysis was used to study the difference of strain and control group. RESULTS:According to 2-DE results, 13 differential protein spots were found by image analysis. Among which 1 spot was new, 7 spots were disappeared, 2 spots were increased, 3 spots were decreased. Whereafter 2 spots were identified initially by mapping peptide mass fingerprinting of protein spots by MALDI-TOF-MS. CONCLUSION: 2 spots were identified from protein database. These results are helpful for further study on the mechanisms of bone remodeling and osteoclast formation under mechanical strain.

AIM: To realize high cell-density fermentation of MAGE3/HSP70 engineered E.coli,and to evaluate the antitumor immunity of MAGE3/HSP70 fusion protein in vivo. METHODS: High cell-density fermentation of MAGE3/HSP70 enginered E. coliwas carried out with constant soluble oxygen and continuous cultivation in self-control fermentor. The mouse was immunized by purified MAGE3/HSP70 fusion protein in E.coli, and the antitumor immunity was detected by ELISPOT and LDH release assay. RESULTS: High cell-density fermentation of MAGE3/HSP70 engineered E.coli was successful, and the expression of MAGE3/HSP70 in fermented bacterial was the same as the flask culture. The results of ELISPOT and LDH release assay showed that MAGE3/HSP70 generated tumor specific cytotoxic T lymphocyte (CTL) to damage tumor cel1. CONCLUSION: High cell- densityfermentation of MAGE3/HSP70 engineered E.coli was realizedand MAGE3/HSP70 fusion protein can generate specific CTL to kill tumor cel1.

Restin, isolated from tumor differentiation cell induced by ATRA, is the novel member of MACE （melanoma antigen encoding gene） family. To obtain some clues of its biological function, this work is going to investigate the restin expression level in variety of tumors and cells. Using the code region of restin labeled with #alpha#-~(32)P-dCTP as a probe, the mRNA expression of restin was examined in multiple tissue northern（MTN） blot membrane and multiple tissue express （MTE）array by Northern blot and dot blot hybridization, respectively. The mRNA expression of restin was examined by in situ hybridization with restin probe labeled with dig in 11 tumor tissues. Northern blot showed that all 12 normal tissues displayed hybrid bands with same location, approximately 1.8 kb. These results suggested that the restin mRNA could not present some splicing variants. Dot blot showed that the restin high expressed in all normal tissues, but less or not expressed in tumor cell lines. In 11 tumor tissues, 8 malignant tumors displayed no expression, but 3 benign tumors displayed lower expression. These results suggested that human restin was rarely expressed in cancer ceil lines, tumor tissues and but ubiquitously expressed in many normal tissues. These results were quite different from the expression of other MAGEs, such as MAGE-A, B, and C. Considering the inducible expression of restin in tumor tissues by ATRA, we deduced that the restin was possibly to be a regulator of cell proliferation and differentiation.

AIM:To obtain recombinant human defensin alpha(HDalpha) and detect its biological activity, so as to facilitate further research.METHODS:The HDalpha gene fragment with hydroxylamine cleavage site was synthesized, and then cloned into pBV220-IL-4 vector to construct pBV220-IL-4-HDalpha. The constructed vector which was confirmed to be correct by sequencing was transformed into E.coli DH5alpha and the IL-4-HDalpha fusion protein was expressed under temperature induction. After fusion protein was cleaved to remove IL-4 by hydroxylamine, purification and renaturation was performed. HDalpha's characteristics were identified by SDS-PAGE and bioactivity detection.RESULTS:After temperature induction, the expressed fusion protein which accounted for about 20% of total bacterial protein existed mainly in the form of inclusion body. After cleaving by hydroxylamine, the purity of obtained HDalpha was about 99.8%. Bacteriostatic test and clone forming test showed that recombinant HDalpha could obviously inhibit the growth of bacteria.CONCLUSION:The recombinant expression plasmid for HDalpha gene has been constructed successfully and obtained engineering bacteria can stably express target protein. Furthermore, techniques of purification and renaturation was set out, which lays the foundation for further functional study and application of HDalpha.

To study the role of all-trans retinoic acid(ATRA) in cell cycle, proliferation, differentiation and apoptosis of breast cancer cell line MCF-7 and the expression of restin gene. Methods: The cell cycle was observed by means of flow cytometry(FCM) , the cell proliferation was detected by MTT assay and the expression of restin mRNA was examined by RT-PCR. Results: Under the treatment by ATRA, the cell growth arrested at G1 phase and the proliferation was inhibited greatly. The level of restin mRNA was found to be up-regulated by ATRA in MCF-7 cell. Conclusion: G1 phase arrest and proliferation inhibition were involved in the process that ATRA induces MCF-7 cell differentiation and apoptosis, and restin maybe play an important role in this process, especially the cell apoptosis.

分子生物学实验技术既是分子生物学的重要组成部分,也是学科藉以发展所不可或缺的工具。对于分子生物学实验课的教学,通过教学实践,对于实验课教学内容的安排、教学形式的选择以及追求怎样的教学效果等问题提出了思考与看法。

AIM: To investigate the effect of Aloe, a kind of traditional Chinese drug, on the rat models of malignant ascites. METHODS: The malignant ascites model of Balb/c rat was induced by S_ 180 cells and then was treated with the different doses of whole leaf, gel and peel of Aloe for 2 weeks. The therapeutic effect was evaluated by comparing the change of rat body mass, protein content in ascite and changes of S_ 180 cell cycle in the rats. RESULTS: The Aloe delayed the rapid increase of the body mass in rat models(P<0.05)and decreased the albumin in ascites (P<0.05). The analysis of cell cycle displayed that the G1 phase cells were decreased and the S phase cells were increased in the Aloe treatment group compared with those in the blank group. CONCLUSION: Aloe showsa great potential in the treatment of malignant ascites.

restin 基因是Zhu等人从全反式维甲酸(all-trans retinoic acid, ATRA)诱导肿瘤细胞分化时克隆得到的一种黑色素瘤抗原相关基因. 前期研究表明, 该基因与细胞周期阻滞有关. 由于p53在细胞增殖调控中占据重要地位, 并且与维甲酸具有诱导关系, 因此本研究试图揭示维甲酸诱导 restin 基因表达是否与p53有关. 将p53转染真核细胞, 研究 restin 与p53的表达关系. 结果表明p53能诱导 restin 基因转录增加. 进一步分析发现, restin 基因5¢端上游约2 kb基因组序列中存在p53蛋白结合位点. 扩增该序列构建荧光报告系统, 检测到该报告系统可以接受维甲酸的诱导调控. 在此基础上, 研究p53对缺失p53结合位点的截短体报告质粒和p53结合位点突变后的报告质粒的作用, 结果显示p53调控restin基因的表达与该基因上游序列区中的p53结合位点无关, 推测p53对 restin 基因的表达调控可能还存在其他分子的相互作用.

目的:为进一步研究在肝癌上过表达的癌相关基因Gankyrin的功能,进行GST-Gankyrin融合蛋白表达载体的构建、原核表达、及抗体制备.方法:采用逆转录PCR(RT-PCR)法从人肝癌细胞系hepG2中扩增Gankyrin Cdna编码区,并将其重组于谷胱甘肽硫转移酶(GST)融合蛋白表达质粒Pgex-4T2中,经酶切、序列鉴定分析后,用该重组质粒转化大肠杆菌DH-5α,经IPTG诱导获得表达,并将Gankyrin蛋白免疫家兔,经过Western blot检测抗Gankyrin抗体的产生.结果:成功构建了Gankyrin原核表达载体,并在大肠杆菌DH-5α中获得表达,Western blot检测证实获得了抗Gankyrin抗体.结论:成功表达了Gankyrin蛋白并制备了其特异性抗体.