七种常见MBL基因单倍型标准质粒的构建

目的 构建7种常见的MBL基因单倍型标准质粒。方法以已确认MBL基因单倍型及基因型的DNA样本为模板．利用SSP-PCR技术扩增出包含MBL基因启动子区及第一外显子的单倍型片段并将其克隆人T载体；用定点突变技术分别将其第一外显子52和57位密码相应的碱基突变；采用PCR和测序进行鉴定。结果以单倍型及基因型已确认的DNA样本为模板，采用SSP-PCR和分子克隆技术获得单倍型HYPA、LXPA、LYOA、LYPA和LYPB重组质粒；以单倍型HYPA及LYOA重组质粒为模板，采用定点突变技术获得单倍型HYPD及LYOC标准质粒。结论构建成功常见的7种MBL基因单倍型标准质粒，为采用SSP-PCR、Real-time PCR等技术分析MBL基因相应的SNP及其单倍型与基因型提供了标准对照。     

Large Envelope Glycoprotein and Nucleocapsid Protein of Equine Arteritis Virus (EAV) Induce an Immune Response in Balb/c Mice by DNA Vaccination; Strategy for Developing a DNA-Vaccine Against EAV-Infection

Equine arteritis virus (EAV) is a member of the Arteriviridae family, that includes lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian haemorrhagic fever virus (SHFV). Equine arteritis is a contagious disease of horses and is spread via respiratory or reproductive tract. The objective of the present study is to evaluate the possibility for developing a model system for prevention horses against an EAV infection by DNA vaccination. A cDNA bank from the RNA of EAV was established. This gene library contains the translation unit of the EAV open reading frames (ORF) 1 to 7. The identity of the cDNA was confirmed by nucleotide sequence analysis. Using this defined EAV cDNA gene library the cDNA sequence of the viral ORFs were molecularly cloned into the corresponding sites of well characterized and powerful expression vectors (pCR3.1, pDisplay, and/or pcDNA3.1/HisC).The capability of these recombinant plasmids expressing the gene products of the individual viral ORFs 3 to 5, and 7 in induction of an immune response in mouse system was investigated. The Balb/c mice (ten mice per assay) were inoculated with the DNA of the constructed expression vectors harboring and expressing the EAV cDNA of the viral ORFs. The Balb/c mice were injected with about 100 g DNA diluted in 100 l PBS. The DNA was injected subcutaneously and into the tibialis cranialis muscle (Musculus gastrocnemius). The mice were boosted 3 to 5 times with the same quantities of DNA and under the same conditions at about two week intervals. Control mice received the same amount of parental expression vectors via an identical route and frequency.The pre- and post-vaccinated sera of the individual animals were screened by neutralization tests (NT). Neutralizing antibodies against EAV were detected when the animals were inoculated with the DNA of the expression vectors harboring cDNA of the EAV ORFs 5 and 7. Highest NT-titers were observed when the animals were administered with the cDNA of ORF 5 and/or with the cDNA of the neutralization determinants of EAV that is located on the N-terminal ectodomain of the gene product of ORF 5 between the amino acid positions 1–121. These results obtained from these studies justified proofing the capability of the EAV cDNA sequences of the viral genes including ORFs 5 and 7 in the autologous animal system horse.     

痢疾杆菌R质粒的检测、消除及药物敏感试验

目的：探讨得疾杆菌的耐药性。方法：采用Ｋ－Ｂ法测定痢疾杆菌的耐药谱,对耐药菌株进行Ｒ闰的检测及接合传递人工诱导痢疾杆功秋稳定Ｌ型,观察Ｒ质粒丢失情况。结果：４５株痢疾杆菌４４经,耐药率９７．８％,其中三重耐药菌株占９３．２％,四重以上耐药菌株占７３．３％。８８．９％（４０／４５）的菌株检测出Ｒ质粒,质粒的接合传递率为６１．４％。痢疾杆菌Ｌ型未见质粒丢失。结论：痢疾杆菌的耐药性十分严重,自由质粒控制     

小鼠白介素-12真核表达质粒的构建及其表达

目的:构建能在真核细胞内稳定表达小鼠白介素-12(mIL-12)的质粒,为进一步研究mIL-12的免疫调节机制及其抗肿瘤作用奠定基础。方法:通过聚合酶链反应(PCR)扩增质粒pORF-mIL-12(Elas-ti),双酶切后将目的片段mIL-12连接到真核表达载体pcDNA3.1( )质粒上,mIL-12受pcDNA3.1( )中mCMV启动子驱动,将mIL-12全长转录至同一mRNA上,对重组质粒进行鉴定后将其转染小鼠Lewis肺癌细胞(LLC)测其表达。结果:PCR成功扩增出mIL-12片段,PCR鉴定、酶切鉴定及测序均证实pcDNA3.1( )-mIL-12质粒构建成功,RT-PCR及ELISA证实重组质粒在mRNA及蛋白质水平均高表达mIL-12。结论:pcDNA3.1( )-mIL-12真核表达质粒构建成功并能在LLC细胞中稳定表达。     

九个苯丙氨酸羟化酶基因单点突变重组质粒的构建和鉴定

目的构建Y154H、R157I、Y206C、G247R、D282G、G346R、S349A、A389G、R400K9个PAH基因突变型重组质粒,为下一步研究这些PAH突变体在哺乳动物细胞中表达蛋白功能奠定基础。方法①选择错义突变位点。结合突变位点所在酶蛋白的结构域(如催化结构域或与辅因子BH4结合位点)及患儿相关临床表型进行了9个突变位点的选择。②构建PAH基因突变型重组质粒。以野生型PAH真核表达载体pcDNA3.0-PAH-wt为模板,应用PrimerPremier5.0软件自行设计了9对突变引物,并在Platin-iumTaqDNA高保真聚合酶的作用下直接利用体外PCR定点突变法构建PAH基因突变型重组质粒。③初筛PAH基因突变型重组质粒。氨苄抗性初步筛选:利用重组基因含氨苄青霉素抗性基因的特点,可筛选出阳性克隆;PCR及酶切技术筛选:根据野生型与突变型靶序列酶切片段的差异进行筛选,其中S349A、D282G、G247R、Y206C、Y154H这5种突变存在MvaⅠ、MvaⅠ、HindⅢ、RsaⅠ、RsaⅠ的天然酶切位点,而R157I、G346R、A389G、R400K与其相应的野生型所在位点周围并不存在天然的酶切位点,需要在目的位点的上/下游设计引物,即人工构建DdeⅠ、HindⅢ、PstⅠ、MvaⅠ酶切位点,然后利用上述限制性内切酶进行突变体的酶切鉴定。④验证PAH基因突变型重组质粒,突变体最终由DNA测序加以验证。结果这9个突变型PAHcDNA序列除特定碱基位点被突变的碱基替换外,其他序列和野生型的完全保持一致。结论成功构建了9个PAH基因突变型重组质粒。体外定点突变技术准确、实用。     

KPC and NDM-1 Genes in Related Enterobacteriaceae Strains and Plasmids from Pakistan and the United States

To characterize the genomic context of New Delhi metallo-β-lactamase-1 (NDM-1) and Klebsiella pneumoniae carbapenemase (KPC), we sequenced 78 Enterobacteriaceae isolates from Pakistan and the United States encoding KPC, NDM-1, or no carbapenemase. High similarities of the results indicate rapid spread of carbapenem resistance between strains, including globally disseminated pathogens.     

DEK基因RNAi慢病毒表达载体的构建及其功能鉴定

目的：构建DEK基因的RNA干扰（ RNAi）慢病毒表达载体,并对其生物学功能进行初步检测。方法采用RNAi技术,根据DEK基因的序列,确定其有效靶序列,合成DEK基因的Oligo DNA,退火形成双链DNA,将其克隆到经BamHⅠ与EcoRⅠ酶切后的小干扰RNA（ siRNA）表达载体PSIH-H1上,产生PSIH-H1-DEK慢病毒载体,筛选阳性克隆并测序鉴定。与3个包装质粒共转染293T细胞,包装成慢病毒后感染乳腺癌细胞ZR75-1,经嘌呤霉素（ puromycin,puro）筛选2周后,收集部分细胞利用实时聚合酶链反应（ real time-PCR,RT-PCR）和Western印迹分别检验DEK在信使RNA （ mRNA ）和蛋白水平的敲低效果,并通过细胞生长实验检测DEK对人乳腺癌细胞系ZR75-1细胞生长的影响。结果 PCR和DNA测序结果证实,DEK siRNA慢病毒表达载体PSIH-H1-DEK构建成功。 RT-PCR和Western印迹结果显示,构建的DEK siRNA可有效抑制DEK基因的表达,并由此建立了敲低DEK的稳定克隆。生长曲线实验表明, DEK siRNA可抑制人乳腺癌细胞系ZR75-1细胞的生长。结论成功构建了DEK基因的RNAi慢病毒表达载体,感染人乳腺癌细胞系ZR75-1细胞后,有效沉默了ZR75-1细胞中的DEK基因的表达,为进一步研究DEK基因在乳腺癌中的作用奠定基础。     

谷胱甘肽过氧化物酶1高表达对β淀粉样蛋白介导PC12细胞损伤的保护作用

目的探讨β淀粉样蛋白(Aβ)所致阿尔茨海默病发病的分子机制,以及谷胱甘肽过氧化物酶1(GPX1)高表达时对其所致细胞损伤的保护作用。方法将PC12细胞转染合人GPX1基因的plncx质粒及空载体plncx质粒,对PCl2、GPXl-PCl2、plncx-PCl2 3组细胞,分别给予Aβ_(25-35)和亚硒酸钠+Aβ_(25-35)2种干预方式,MTT法检测细胞的存活情况,免疫细胞化学法观察磷酸化环磷酸腺苷反应元件结合蛋白(pCREB)的表达情况。结果 PCl2组与plncx-PCl2组细胞存活率比较,差异无统计学意义(P>0.05);GPXl-PC12组较plncx-PCl2组细胞存活率明显增高(P<0.01);Aβ_(25-35)干预后,plncx-PC12组和GPX1-PC12组pCREB蛋白阳性率明显下降,plncx-PC12组较GPX1-PCl2组下降更明显(P<0.05)。亚硒酸钠+Aβ_(25-35)干预后,plncx-PC12组和GPX1-PC12组pCREB蛋白阳性率明显上升,GPX1-PC12组较plncx-PC12组增高更明显(P<0.01)。结论 GPX1基因高表达对Aβ_(25-35)所介导的PCl2细胞损伤有明显保护作用;Aβ_(25-35)可下调pCREB在PCl2细胞中表达,而GPXl与pCREB共同参与保护PCl2细胞,减少Aβ_(25-35)所引起的细胞损伤。