EB病毒VCA基因片段在毕赤酵母中的表达及临床应用

目的 探讨EB病毒(Epstein-Barr vires,EBV)壳抗原VCA-BALF4(S)和VCA-BFRF3重组蛋白在鼻咽癌血清学诊断中的应用.方法 以EBV DNA为模板,采用PCR法扩增目的 基因片段BALF4(S)和BFRF3,与毕赤酵母表达载体pPICZaA连接,转化酵母菌GS115,甲醇诱导重组蛋白表达.表达产物经SDS-PAGE、免疫印迹法鉴定后,纯化目的 蛋白作为包被抗原,制备ELISA试剂检测鼻咽癌患者和正常人群EBV-IgA抗体.结果 在毕赤酵母菌中成功高效地表达了分泌型的VCA-BALF4(S)和VCA-BFRF3重组蛋白,相对分子质量分别为37×10~3和18×10~3,免疫印迹证实目的 带均有免疫原性,目的 蛋白经纯化后作为包被抗原检测鼻咽癌患者和健康对照的敏感度和特异度分别为71.0%和91.8%(VCA-BFRF3)、88.7%和96.4%[VCA-BALF4(S)].结论 毕赤酵母系统高效分泌表达了VCA-BALF4(S)和VCA-BFRF3重组蛋白,对两种重组抗原在鼻咽癌血清筛选中的诊断价值进行了初步评估,获得了在鼻咽癌筛选中有一定应用价值的pPICZa-BALF4(S)生产酵母工程菌株.     

外源基因在巴斯德毕赤酵母中的表达

酵母是单细胞真核生物，既具有类似原核生物的生长特性，又具有一般真核生物的细胞生物学特性。巴斯德毕赤酵母是新近发展起来的新型表达系统，许多有应用价值的外源基因成功地在其中表达，使其日益受到关注。本就毕赤酵母的生物学特性、表达系统的构建和分泌蛋白的翻译后修饰等方面进行综述。     

汉坦病毒部分S基因在毕赤酵母中的分泌表达

目的 构建并表达汉坦病毒(HV)Z10株(HV-Z10)S基因蛋白编码区前300 bp核苷酸序列,研究该基因在毕赤酵母(Pichia pastoris)中的表达及其产物的生物活性.方法 根据HV-Z10 S基因前300 bp(S300)的核苷酸序列,按照编码氨基酸的密码子转换成酵母偏爱的形式,设计合成8条引物,通过连续PCR,获得人工合成Z10株S基因前300 bp的基因序列SP300,经测序,SP300与S300的核苷酸相似性为76.2%,但编码的氨基酸序列完全一致.将SP300克隆到酵母穿梭载体pPICZaA,构建含α-factor分沁信号肽的重组表达载体pPICZaA-SP300.将pPICZaA-SP300、pPICZaA-S300化学法转化酵母GS115菌株,筛选重组转化子.结果 重组了SP300与S300的酵母转化子经甲醇诱导,表达出重组蛋白rNP300及rN300,SDS-PAGE显示相对分子质量为12×10~3左右.经ELISA检测及Westem Blot分析,表达产物能与抗汉坦病毒抗体起免疫反应.结论 SP300和S300基因在毕赤酵母中获得分泌表达,使用酵母偏爱密码子的SP300基因在毕赤酵母中的表达量与S300的表达量基本一致,表达量在诱导24 h后最高.     

外源基因在巴斯德毕赤酵母中的表达

酵母是单细胞真核生物 ,既具有类似原核生物的生长特性 ,又具有一般真核生物的细胞生物学特性。巴斯德毕赤酵母是新近发展起来的新型表达系统 ,许多有应用价值的外源基因成功地在其中表达 ,使其日益受到关注。本文就毕赤酵母的生物学特性、表达系统的构建和分泌蛋白的翻译后修饰等方面进行综述     

mTLR-2基因的克隆及其在毕赤酵母中的表达

目的 :克隆mTLR 2基因 ,并在毕赤酵母中的表达其融合蛋白。方法 :采用RT PCR从鼠肝脏中扩增mTLR 2全基因 ,并将其克隆到T载体中 ,测序验证。将目的基因编码序列插入毕赤酵母表达载体pPICZαC中 ,构建重组质粒 ,并转化毕赤酵母。重组酵母以PCR、RT PCR验证 ,表达的重组蛋白用SDS PAGE和Westernblot进行分析。结果 :克隆了mTLR 2全基因(AY179346 ) ,与已发表的mTLR 2基因的同源性为 99.84 %。构建了重组表达质粒pPICZ mTLR 2。SDS PAGE和Westernblot分析显示 ,在相对分子质量 (Mr)为约 970 0 0处出现 1条特异性蛋白带 ,且能与兔抗mTLR 2抗体发生反应。结论 :克隆了mTLR 2全基因 ,并在毕赤酵母中获得表达。     

抗人肝癌单克隆抗体嵌合Fab在巴氏毕赤酵母中的高效分泌表达

目的:通过分步整合,用巴氏毕赤酵母表达抗人肝癌单克隆抗体(mAb)HAb18的嵌合Fab(cFab)片段。方法:将抗人肝癌mAbcFab/HAb18基因的原核表达载体pET32a/cFab中的CL和Fd段基因,分别亚克隆到酵母表达载体pPIC9K和pPICZαA中,构建重组质粒pPIC9K/CL和pPICZαA/Fd并测序鉴定。将重组质粒pPIC9K/CL和pPICZαA/Fd分步整合到酵母菌GS115的染色体上,经G418和Zeocin筛选高拷贝的转化子及鉴定Mut表型后,用含5mL/L甲醇的培养基诱导表达。结果:成功地表达抗人肝癌mAbHAb18的cFab,表达水平为26mg/L。Westernblot鉴定证实,表达产物具有良好的与HAb18结合的活性和特异性。结论:cFab/HAb18在巴氏毕赤酵母获得表达,为对其进一步大规模的生产和临床应用奠定了基础。     

人乙醛脱氢酶2基因在毕赤酵母SMD1168中的表达研究

目的得到一株产人乙醛脱氢酶2(acetaldehyde dehydrogenase-2,ALDH2)的重组毕赤酵母菌株,优化其发酵条件以满足生产和科研需求。方法将ALDH2基因整合到质粒pPIC9K,构建重组表达载体pPIC9K-ALDH2,将重组表达质粒pPIC9K-ALDH2经电转化到毕赤酵母SMD1168中,转化子经MD平板筛选后,用G418筛选多拷贝重组子,测序鉴定。通过改变诱导温度、pH、初始OD值、甲醇浓度等诱导条件,对SMD1168(pPIC9K-ALDH2)发酵的条件进行优化。结果ALDH2cDNA整合到毕赤酵母基因组中,得到SMD1168(pPIC9K-ALDH2);获得最佳的诱导表达优化条件:在28℃、pH=7.0、诱导初始OD600=12、每隔24h添加1.5%甲醇、转速为300r/min,发酵所产生的人ALDH2酶活可达到0.115U/ml,相对较高。结论以蛋白酶缺陷型毕赤酵母SMD1168为宿主,分泌表达质粒pPIC9K为载体,能够成功构建重组子SMD1168(pPIC9K-ALDH2)。     

EGF在毕赤酵母中的表达及活性测定

目的高效表达和纯化有活性的EGF蛋白。方法将含有表皮生长因子(EGF)基因的质粒pAO815通过LiAc法转入毕赤酵母细胞中,筛选在MM平板上生长缓慢的Muts型酵母工程菌,甲醇诱导基因表达产生EGF,柱纯化后利用SDS-PAGE检测到EGF的生成,MTT法及新生小鼠皮下法分别检测活性。结果 SDS-PAGE电泳检测EGF成功表达和纯化,MTT法测定EGF生物活性约为2.8×106IU/mL。结论本研究构建的工程菌株能高效表达EGF,且纯化的EGF具有与天然EGF同样的体外和体内活性。     

乙型肝炎病毒C基因在毕赤酵母中的表达

目的:研究乙肝病毒核心(C)基因在毕赤(Pichia pastoris)酵母中的表达,以期获得高效表达的具有良好免疫反应性和特异性的重组乙肝病毒核心蛋白(HBcAg)。方法:采用PCR法从含HBV全基因序列的质粒pHBV1中扩增C基因,亚克隆到pGEM-T载体中,经DNA序列分析后将目的基因定向克隆到酵母表达载体pPIC9中,构建重组质粒pPIC9-cAg。然后用电转法将重组质粒转化入酵母菌GS115,0.5%甲醇诱导表达。采用SDS-PAGE、Western blot和ELISA法对表达产物进行分析。结果:限制性内切酶酶切和DNA序列分析证实HBV C基因已正确克隆到酵母表达载体pPIC9中;SDS-PAGE结果显示重组HBcAg在毕赤酵母细胞中表达;ELISA及Western blot分析表明,表达产物具有良好的免疫反应性和特异性,重组HBcAg的滴度可达1∶12 800。结论:成功构建了pPIC9-cAg重组质粒,并在毕赤酵母中高效表达了具有良好免疫反应性和特异性的重组HBcAg,为进一步研制抗-HBc诊断试剂盒奠定了基础。     

表达HIV-1 gag-gp120嵌合基因酵母工程菌的构建及表达条件的优化

目的：构建表达HIV-1gag-gp120嵌合基因的酵母工程菌，并优化表达条件。方法：将gag-gp120嵌合基因插入到酵母表达载体pHILS1中，构建了表达质粒pHILGP。线性化质粒电转化毕赤酵母菌GS115后进行整合，通过PCR及表达产物的SDS-PAGE和ELISA等方法筛选 阳性酵母工程菌，并优化表达条件。结果：成功构建表达融合蛋白的酵母工程菌，表达量为13%左右。表达蛋白能与HIV-1阳性血清发生反应，但其相对分子质量（Mr）比预计计算的值要小。最佳表达条件为：BMMY培养基，85%溶解氧，培养时间3d，1%甲醇诱导浓度。结论：表达的融合蛋白具有很好的抗原特异性，存在于上清中，这有利于目的蛋白的分离纯化。     

MRSA耐药相关TG-TPase嵌合基因的原核表达与纯化

目的 设计、构建耐甲氧西林金黄色葡萄球菌(methicillin resistant Staphylococcus aureus,MRSA)耐药相关TGTPase嵌合基因,进行原核表达、纯化探索,为进一步利用其酶学活性建立抑制剂筛选系统奠定基础.方法 通过引物设计,从盐平板法筛选的MRSA菌株中分别克隆PBP2的TG基因片段和PBP2a的TP片段,分别克隆入T载体,对阳性重组子进行酶切/再连接,构建TG-TPase嵌合基因,经核苷酸测序鉴定正确的嵌合基因再亚克隆到pET22b,构建表达载体,转化Rosetta(DE3)plysS,用LPTG进行诱导表达,并对表达的蛋白进行SDS-PAGE、质谱和Westem blotting鉴定.小量发酵重组菌,对嵌合基因表达产物进行初步纯化.结果 从13株临床分离的金葡菌中筛选出2株高耐药性MRSA菌株,用PCR法从中成功地克隆到青霉素结合蛋白PBP2的TG片段和PBP2a的TP片段,构建了TG-TPase嵌合基因及其原核表达载体,并在大肠埃希菌中表达,产量达菌体总蛋白的43%.融合蛋白纯化分析表明,嵌合蛋白以包涵体形式存在,在变性条件下经Ni-NTA亲和柱纯化,纯度达90%以上.结论 成功设计并构建了耐药性金葡菌TG-TPase嵌合基因及其重组表达工程菌,为进一步利用其酶学活性进行抗耐药性金葡菌抑制剂的筛选奠定基础.     

Management of methicillin-resistant Staphylococcus aureus infections

This review addresses selected aspects of the management of severe healthcare-associated infections due to methicillin-resistant Staphylococcus aureus (MRSA), including the limitations of current therapy, potential alternative agents, new therapeutic options, clinical approaches to MRSA bacteraemia/endocarditis and ventilator-associated pneumonia, and strategies to improve outcomes in patients with severe MRSA infections.     

Heterogeneity of methicillin-resistant Staphylococcus aureus isolated in Norway

Our objective was to look for differences in susceptibility patterns between Norwegian and imported methicillin-resistant Staphylococcus aureus (MRSA) strains. All MRSA isolates from the participating hospitals (87 isolates from 81 patients) throughout the period 1994–98 were examined, to study the clonal distribution of MRSA isolated in Norway and to identify any epidemic clones among the isolates. We found that imported isolates were resistant to an average of 5.6 antibiotics, while Norwegian isolates were resistant to an average of 2.6 antibiotics. MRSA isolates imported to Norway are more often multiresistant than domestic isolates. MRSA isolates in Norway show a striking diversity. Epidemic clones are present, but no single clone is predominant.     

Molecular genetics of methicillin-resistant Staphylococcus aureus

A large and growing proportion of Staphylococcus aureus clinical isolates are methicillin resistant, and are resistant to practically all beta-lactam antibiotics. Methicillin-resistant S. aureus (MRSA) strains harbor mecA, which is carried by a unique mobile genetic element, staphylococcal cassette chromosome mec (SCCmec) integrated into the S. aureus chromosome. The mecA gene encodes a methicillin-insensitive transpeptidase, the production of which confers resistance to otherwise inhibitory concentrations of beta-lactam antibiotics. Several distinct clones have been identified among MRSA that apparently have been generated by integration of distinct types of SCCmec. While MRSA are primarily nosocomial pathogens, recent observations indicate that other MRSA clones are colonizing a significant proportion of healthy individuals in the community as well. Community-acquired MRSA (C-MRSA), may become a new threat to humans, and international cooperation of researchers and clinicians will be of cardinal importance in addressing this problem.     

Prevalence of methicillin-resistant Staphylococcus aureus in eight African hospitals and Malta

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious therapeutic problem worldwide, and its frequency in most African countries has not been reported. This study was aimed at determining the prevalence and antibiotic susceptibility patterns of MRSA in eight large hospitals (>500 beds) in Africa and Malta, from 1996 to 1997. Susceptibility to methicillin (oxacillin) and to other drugs was determined by E test (AB Biodisk, Solna, Sweden) on a total of 1440 clinical isolates of S. aureus . Methicillin resistance was detected in 213 (15%) of the 1440 isolates tested. The rate of MRSA was relatively high in Nigeria, Kenya, and Cameroon (21–30%), and below 10% in Tunisia, Malta, and Algeria. All MRSA isolates were sensitive to vancomycin, with MICs ≤ 4 mg/L. The isolates were also highly sensitive to ciprofloxacin, except in Kenya, Morocco, and Tunisia, where relative resistance to this drug was noted. Susceptibility to rifampin and fusidic acid seems to be correlated with the clinical use of these compounds. Only 46% of 59 MRSA strains analyzed were susceptible to rifampin, fusidic acid, and ciprofloxacin. The majority (> 60%) of MRSA strains were multiresistant. There is a need to maintain surveillance and control of MRSA infections in Africa.     

Characterisation of methicillin-resistant Staphylococcus aureus isolates from dogs and their owners

Ten methicillin-resistant Staphylococcus aureus (MRSA) isolates from healthy owners and their pets were characterised by susceptibility testing, staphylococcal chromosome cassette (SCC)mec and agr typing, and detection of the Panton-Valentine leukocidin (PVL) genes. Two human and three dog isolates harbouring SCCmec type III appeared to be of hospital origin. The five remaining isolates carried SCCmec type IV, with three being multidrug-resistant. One type IV isolate was PVL-positive and a prototypic agr type 3, typified by strain MW2. This is the first report of this type in association with nasal carriage. Drug resistance may be increasing among community isolates of MRSA.     

Decolonization of methicillin-resistant Staphylococcus aureus using oral vancomycin and topical mupirocin

The objective of this study was to assess the efficacy and safety of a short course of oral vancomycin and intranasal mupirocin ointment in the eradication of methicillin-resistant Staphylococcus aureus (MRSA) colonization. During an outbreak of MRSA, the colonized subjects received oral vancomycin and topical mupirocin. They were screened for MRSA 1, 3, 6 and 12 months after decolonization. A questionnaire was developed to evaluate the side-effects of oral vancomycin. Thirty-five subjects were treated. Clearance was achieved in all cases, in 24 (69%) subjects after one course of therapy. Twenty-eight (80%) subjects experienced some side-effects, including six (17%) who did not tolerate oral vancomycin. Although oral vancomycin, in combination with topical mupirocin, is effective in the elimination of MRSA colonization, there is a need for further studies to confirm our results and to evaluate the safety of oral vancomycin.     

Costs of healthcare-associated methicillin-resistant Staphylococcus aureus and its control

Methicillin-resistant Staphylococcus aureus (MRSA) clones have caused a huge worldwide epidemic of hospital-acquired infections over the past 20–30 years and continue to evolve, including the advent of virulent community strains. The burden on healthcare services is highly significant, in particular because MRSA has not replaced susceptible staphylococcal infection but is an additional problem. Treatment strategies for MRSA are suboptimal and compromise the care of patients. MRSA is associated with serious morbidity and mortality, both within and without hospitals. Although the literature on the costs of MRSA and its control is suboptimal, it is clear that the control of MRSA is highly desirable and likely to be cost-effective. Any compromises in control are likely to be false economies.