目的 Sj16蛋白是从日本血吸虫分泌物中分离出的一种具有免疫调节功能的蛋白质,具有诱导器官移植免疫耐受的潜力.但是,在用传统方法合成时,其表达量低,可溶性较差,影响了其生物活性.该实验拟优化Sj16AA的合成方法,为进一步研究其功能提供条件.方法 在Sj16蛋白的DNA序列基础上,进行特定位点的突变修饰,同时针对大肠杆菌表达系统优化密码子.将修饰后的Sj16AA基因定向克隆入pMal-c2X质粒,转入BL21(DE3)菌表达,摸索不同条件下Sj16AA的表达情况.结果 成功制备目的基因片段与载体的连接产物pMal-c2X-Sj16M,转化后的BL21/DE3菌经IPTG诱导可表达与MBP融合的蛋白(约58 kDa),抗MBP抗体能特异性识别该蛋白.结论 相较于传统合成方法,经优化的Sj16AA蛋白表达量较高,可溶性较好,能够更好地应用于器官移植免疫相关的基础和临床研究.

Chlamydiae are very important pathogens which could cause several types of diseases in human, but little is known about its pathogenic mechanism. In order to elucidate host inflammatory response and the signal pathway induced by Chlamydial lipoproteins, the predicted lipoproteins of Chlamydia trachomatis were tested for their ability to induce the release of proinflammatory cytokines by mouse macrophages or human TLR (Toll-Like Receptor) expressing cell lines. The results showed that recombinant proteins of C. trachomatis D381, D541, D067, and D775 displayed a strong ability to induce the release of IL-8 in TLR expressing cell line. The signal pathways involved TLR1/2 and TLR2/CD14 but not TLR4. Moreover, except D067, the proinflammatory cytokine induction by D381, D541, and D775 required the thioacylation site (cysteine) for lipid modification and the induction was through MyD88-mediated pathway. Our data supported that lipoproteins played a vital role in pathogenesis of C. trachomatis-induced inflammatory responses via TLR pathway. It was the first study to characterize other chlamydial lipoproteins after identifying the role of MIP (D541) on pathogenesis of Chlamydial diseases.

目的 构建HW11c40突变体,寻找合适的表达系统表达HW11c40突变体和HWTX-Ⅺ(虎纹捕鸟蛛毒素-Ⅺ),解决HWTX-Ⅺ来源问题和HW11c40改造后毒素难于获取的难题,为合适HW11 c40突变体的筛选和治疗急性胰腺炎高效新药的研制奠定基础. 方法 用PCR定点突变的方法突变HW11c40相关氨基酸残基,构建HW11c40突变体.HW11c40突变体和HWTX-Ⅺ基因经PCR扩增后插入到pET-40b(+)载体,基因5'端插入肠激酶切割位点,再重组到大肠埃希菌BL21 (DE3)进行原核周质表达,获得相应的融合蛋白并进行SDS-PAGE鉴定,用镍柱纯化融合蛋白.融合蛋白用肠激酶酶切,释放出的目的蛋白用Tricine-SDS-PAGE进行鉴定,用镍柱分离目的蛋白,进行高效液相色谱进行进一步分离纯化后作质谱鉴定.对HW11c40突变体和HWTX Ⅺ的表达时间、温度和IPTG浓度进行优化. 结果 成功获得HW 11 c40的4个突变体,并正确构建HW11c40突变体和HWTX-Ⅺ的原核表达载体.经SDS-PAGE和Tricine-SDS-PAGE鉴定,重组质粒转化菌在无IPTG诱导的情况下成功表达所有HW11c40突变体和HWTX-Ⅺ的融合蛋白,经肠激酶酶切均获得对应的目的蛋白,其中HWTX-Ⅺ表达量为3.4 mg/L. 结论 成功构建出HW11c40突变体和HWTX-Ⅺ的重组质粒并在原核细胞内高效表达出相应的目的蛋白,开辟了基因工程表达HWTx-XⅪ的新途径,同时解决了 HW11c40改造后毒素获取难题,突破了整个系列研究的瓶颈,为突变体的筛选及治疗急性胰腺炎高效新药的研制奠定了基础.

Objective:To observe the effect of Pidotimod and Thalidomid to enhance the immune response and protective immunity induced by the epitope Vaccines From W2b2a of Toxoplasma gondii in mice.Methods:Mice were immunized intramuscularly with Pidotimod and pcDNA3-W2b2a,thalidomide and pcDNA3-W2b2a,respectively,then the immune response and the survival time of mouse attacked by Toxoplasma gondii were observed.Results:After the immunization,the level of IFN-γin sera of mice inculated with pcDNA3-W2b2a and Pidotimod,pcDNA3-W2b2a and Freund adjuvant,pcDNA3-W2b2a and Thalidomid were significantly higher than pcDNA3-W2b2a (all P<0.05).After the immunization,IgG,CD4+/CD8+T cell ratio ,proliferation of T cell induced by pcDNA3-W2b2a and Pidotimod,pcDNA3-W2b2a and Freund adjuvant were higher than pcDNA3-W2b2a,pcDNA3-W2b2a and thalidomid ( all P<0.05).After challenged with highly virulent tachyzoites,the mean survival time in immunized groups were significantly longer than control group (all P<0.05).Conclusion:Pidotimod ,Thalidomid adjuvant can increase protective immunity of epitope Vaccines From W2b2a of Toxoplasma gondii in mice.

目的 探讨黄芪对弓形虫wx2b2a表位疫苗免疫小鼠后抗急性弓形虫感染的免疫保护作用.方法 将小鼠随机分成pcDNA3-W2b2a组、pcDNA3-W2b2a+黄芪治疗组、pcDNA3+黄芪治疗组、黄芪治疗组、pcDNA3及生理盐水对照组.将弓形虫pcDNA3-W2b2a肌注免疫pcDNA3-W2b2a组、pcDNA3-W2b2a+黄芪治疗组小鼠3次,末次免疫后4周,每组小鼠腹腔注射102个速殖子,感染后2d起,pcDNA3-W2b2a+黄芪治疗组、pcDNA3+黄芪治疗组及黄芪治疗组小鼠给予黄芪75mg/d灌胃治疗,连续用药7d,用ELISA法测定免疫前、末次免疫后4周、感染后6d小鼠血清IgG水平及免疫前、末次免疫后2周、感染后4、6、8d小鼠IFN-γ、IL-18水平,并观察弓形虫感染后小鼠的生存时间.结果 pcDNA3-W2b2a免疫小鼠后血清IgG抗体水平均高于其它组(P＜0.05),末次免疫后4周,感染后第6 d pcDNA3-W2b2a、pcDNA3-W2b2a+黄芪治疗组小鼠特异性IgG抗体水平无显著性差异(P＞0.05);末次免疫后2周、感染后4d、6d、8 d pcDNA3-W2b2a+黄芪治疗组小鼠血清IFN-γ水平分别高于其它组(P＜0.05);感染后各组小鼠血清IL-18水平持续上升,但感染后8d,黄芪治疗后小鼠血清IL-18水平显著低于pcDNA3和生理盐水对照组(P＜0.05);小鼠RH株速殖子感染后,pcDNA3-W2b2a+黄芪治疗组小鼠存活时间明显长于pcDNA3-W2b2a组、pcDNA3+黄芪治疗组及黄芪治疗组(P＜0.05).结论 黄芪可增强弓形虫wx2b2a表位疫苗免疫小鼠后抗急性弓形虫感染的免疫调节作用.

目的 探讨黄芪多糖(Astragalan)、香菇多稽(Lentinan)增强弓形虫wx2b4a表位疫苗刺激机体产生免疫应答和保护免疫的效果.方法 将黄芪多糖、香菇多糖分别与弓形虫wx2b4a表位疫苗混合肌注免疫小鼠,ELISA法检测小鼠血清中IgG抗体水平,pcDNA3-W2b4a刺激下培养各组小鼠脾脏淋巴细胞,ELISA法检测IL-2、IFN-γ分泌水平,CCK-8法检测免疫鼠脾细胞增殖活性,并观察其受到弓形虫攻击感染后的生存时间.结果 各免疫组小鼠血清IgG抗体水平显著高于对照组(P＜0.05),且pcDNA3-W2b4a+黄芪多糖组要高于pcDNA3-W2b4a+香菇多糖组(P＜0.05);pcDNA3-W2b4a+黄芪多糖组、pcDNA3-W2b4a+香菇多糖组小鼠脾细胞IL-2、IFN-γ水平高于pcDNA3-W2b4a组(P＜0.05);各免疫组T细胞增殖活性与对照组比较明显增强(P＜0.05),且pcDNA3-W2b4a+香菇多糖组要高于pcDNA3-W2b4a+黄芪多糖组;小鼠攻击试验表明,pcDNA3-W2b4a+黄芪多糖组、pcDNA3-W2b4a+香菇多糖组小鼠存活时间明显长于对照组和pcDNA3-W2b4a组(P＜0.05).结论 黄芪多糖、香菇多糖可增强弓形虫wx2b4a表位疫苗产生免疫应答,并显示较好的免疫保护效应.

本文报告两例牛带绦虫感染病例,其中一例国内感染,另一例系非洲埃塞俄比亚输入性感染.2例患者均采用槟榔-南瓜子法进行驱虫治疗.病例1驱虫治疗后在粪便中检获部分孕节片,查找有完整头节.病例2治疗后排出一活的完整虫体,显微镜下可见头节近方形,无顶突和小钩.治疗后2～3个月电话随访,两例患者均痊愈.

大部分医学生毕业后不愿意从事基础医学工作,主动报考基础医学专业研究生的人数呈快速递减之势、生源质量不断下滑,导致基础医学人才储备严重不足,已成为制约我国基础医学可持续发展的重要瓶颈。为此,从2009年开始,尝试在五年制临床医学专业学生中开办基础医学强化班,以提高学生对基础医学的兴趣,选拔培养有潜力的学生将来从事基础医学教学与研究。强化班已持续开办4届,现就其培养目标、管理模式、特色课程以及办班进展情况进行探讨。

Human Parasitology is a course designed for medical undergraduate students.How to adapt to the development trend of translational medicine,grasp the innovation opportunities that provided by translational medicine,on the base of deeply study and understand the basic contents and innovation about translational medicine,and how to take use of a series of effective measures to improve the parasitology teaching situation of medical undergraduate in order to enhance the quality of teaching are still a research topic which required carefully thought and collaborative research.This article explores a number of issues that the human parasitology translational medicine involves.

Kunitz型胰蛋白酶抑制剂属于典型丝氨酸蛋白酶抑制剂,其生理功能至少包括了保护储存蛋白、调节内源蛋白酶的活性、保护个体免爱昆虫和病原体的侵害、抑制癌细胞侵袭扩散、抗炎、抗真菌等作用.并已经应用到了抗虫性转基因植物和临床疾病治疗中.

This article introduced the think and aim of construction of experimental central of cell and molecular biology in cen-tral south university,and the contents of construction in the matter of hardware and environment,experimental teaching staff and refor-mation and innovation of experimental teaching system and so on.Four aspects from optimizing the contents of experimental teaching,establishing the mode of opening experimental teaching,establishing the shared platform of experimental technique and establishing the quality guarantee system of experimental teaching were mainly introduced.It is supplied referenced mode for the construction of the ex-perimental teaching demonstration center in home.

Objective To construct a cDNA library of a gene coding for toxin in the spines of the slug moth(Lepidoptera: Eucleidae).Methods Total mRNA was extracted from the slug moth's spines using a SMARTTM cDNA Library Construction Kit from Clontech Laboratories.The second chain was replicated,linked to the vector,and then transformed into XL1-Blue competent cells.Mono-clones with inserts larger than 300 bp were sequenced after screened with PCR.Results The cDNA library was successfully constructed.Target fragments 250-1 000 bp in size were preliminarily identified.Conclusion A cDNA library of a gene coding for the toxin in the spines of a slug moth was successfully constructed,and the entire sequence of the gene coding for this spine toxin was obtained.This should provide a basis for future research on the mechanism of the toxin's pathogenicity.

In recent years we have explored three modes of early scientific research training on 8-year-system medical students:open-title training,probationary training and one-to-one training.The first type of training is that the tutors prepared topics for all of 8-year-system medical students.Students should consult references and apply for grant by themselves,then tutors gave an evaluation of the innovation and feasibility.Probationary training meaned that teachers organized all of students to visit laboratories and learn to choose the topics according to what they are interested in.One-on-one training is that students had to look for their supervisors voluntarily and submit their own original subjects.After supervisors affirmed innovation and feasibility of the subjects,students should complete it under the direction of the supervisor.After some practices,we found that the first type could exert all students' initiation and encourage them to explore scientific issues.The second type of training enabled students to have initial perceptions of scientific research and aroused their research interest.The last pattern could guide students to do some early scientific reseach by taking advantages of the supervisors and disciplines and provide platform to those students who own scientific research potential,also could create good conditions for cultivating students' creative and practical ability.In a word,the three models of the early scientific research training can improve the medical students' research capacity,it also lay a solid foundation for us to train them into high-quality and internationally competitive medical talents in the future.

刚地弓形虫属于真球虫目弓形虫科,作为一种专性细胞内寄生原虫,几乎可感染包括人类在内的所有哺乳动物、鸟类和爬行类等动物.它是人或动物流产、死胎、弱智儿等同产期疾患病和导致感染者发生眼科疾病及脑神经疾病的病原体;同时,它又是一种机会致病寄生虫,在机体免疫能力低下如艾滋病患者、器官移植及恶性肿瘤患者中,可引起严重的并发症甚至死亡.近年来,随着艾滋病发病率逐年上升和宠物饲养的增多,弓形虫病发病率也随之升高,已成为一个严重的、世界性的公共卫生问题.然而,治疗该病的传统药物如乙胺嘧啶、磺胺类制剂等复发率高、根治率低、疗程长,至今尚未找到理想的治疗药物.随着生命科学技术的发展和应用,人们从分子水平上弓形虫速殖子侵入宿主细胞的分子机制有了越来越深入的认识.人们发现弓形虫是一种对嘌呤、胆固醇和氨基酸等重要代谢物质的专性胞内寄生原虫,其虫体内合成这些营养物质的酶类存在种属特异性,从而为寻找有效的药物作用靶位点成为可能,现就该领域的研究进展概述如下.

Objective To clone and express peptides from the spider Ornithoctonus huwena and to lay the foundation for further research into their biological functions.Methods A venom gland cDNA library was constructed for the spider O.huwena and random clones were sequenced.Genes encoding peptides were cloned,eukaryotic expression plasmids were constructed,and Saccharomyces cerevisiae strain S78 was used as a secretory expression system.The expression yield was identified by Tricine SDS-PAGE.Results A novel gene,named HWTX-XIVa2,encoding a cysteine-rich peptide was cloned.The recombinant plasmids were transformed into S.cerevisiae S78.The molecular mass of the expressed peptide was about 6.5 ku.Conclusion The construction and screening of the venom gland cDNAs library is a promising method with regard to the efficient characterization of potential peptide toxins.Expression in S.cerevisiae S78 is a reliable method of obtaining peptides.

In the light of present educational situation,to carry out a reform in university education for conforming to the requirements of the times is a natural trend.Therefore,the methods and means of teaching should continuously satisfy the demand of High-speed development in the information age.Informational teaching models,just as multimedia,have been widely applied and extensively spread in teaching activities of modern universities.However,there are still a wide variety of disputes and misunderstandings in how to correctly deal with and utilize the ways and means of multimedia teaching and the disputes and misunderstandings are no longer simple accumulation of some cases.This article is to comprehensive evaluate practical application of multimedia in undergraduate teaching of Medical Parasitology,a modern basic medical curriculum,affirm the advantages of multimedia teaching,objectively describe and descript its dilemma by combining my own cognition accumulated from process of teaching,make a few more feasible solutions and, by these ways,probe into a multimedia teaching strategy.

The micronemal protein MIC3 of Toxoplasma gondii plays a predominant role in the early phase of the invasion process. In this research, the expression and location of protein MIC3 and fusion protein EGFP-MIC3 were observed in host cells and in T. gondii respectively. Protective experiments of DNA vaccine pcDNA3-MIC3 (pMIC3i) in animals showed that the vaccine could significantly prolong the survival time of the mice challenged by virulent RH strains of T. gondii. All mice vaccinated with plasmid pcDNA3-MIC3 have been elicited specific humoral immunity and cellular immunity. The cellular immune response was associated with the increase of the CD4(+) and CD8(+) T lymphocytes and the decrease of the CD4(+)/CD8(+) T lymphocyte ratio evidently. It indicated that the mic3 DNA vaccine could stimulate the host resisting Toxoplasma mainly by the way of CD8(+)CTL cells. In conclusion, a potent DNA vaccine pcDNA3-MIC3 could elicit a strong specific immune response and induce effective protection against T. gondii challenge in Kunming mice, suggesting that mic3 is a potential vaccine candidate against toxoplasmosis.

[Objective] To construct a overexpressed recombinant plasmid marked with YFP of wx2 gene from Toxoplasma gondii,and provide a tool for the researching of the gene function of new gene wx2.[Methods] The wx2 opening reading frame(ORF) was amplified with two specific primers removed stop code.The ORF and plasmid p tubYFP-YFP/sagCAT were digested with BglII and AvrII respectively and the ORF was ligated into the ptubYFP-YFP/sagC-AT at the YFP-1 site.The recombinant vector pwx2-tub-YFP sag/CAT was characterized by PCR,restriction enzyme digestion,and sequencing analysis.[Results] The expected ORF,558 bp long,was amplified by PCR,and inserted into plasmid ptubYFP-YFP/sagCAT.PCR,restriction enzyme digestion and sequencing analysis showed that pwx2-tub-YFP sag/CAT contained wx2 ORF with the right orientation.[Conclusion] Overexpressed recombinant plasmid wx2-tub-YFP sag/CAT marked with YFP of a new gene wx2 is successfully constructe.