人呼吸道合胞病毒RNA聚合酶复合体蛋白N、P的表达鉴定

本研究根据编码A亚型人呼吸道合胞病毒(HumanRespiratorySyncytialVirus,HRSV)核壳体蛋白(Nucleocapsidprotein,N)和磷蛋白(phosphoprotein,P)的基因序列,各设计一对特异性的引物,应用RT-PCR技术,从感染HRSV的HEp-2细胞中扩增获得n和p的基因片断,克隆至真核表达载体pcDNA3.1( )。获得的重组质粒通过脂质体Lipofectamine2000转染COS-7细胞,72h后再用Westernblot鉴定蛋白的表达。结果显示真核表达载体pcDNA3.1( )/N和pcDNA3.1( )/P的限制性内切酶分析结果与预期一致,基因序列分析显示没有发生无义突变,利用蛋白印记方法也检测到了N和P的特异性条带。于是我们认为成功构建了含有HRSVN和P编码基因的真核表达载体,在真核细胞内能顺利表达。为进一步开展HRSV反向遗传学等研究奠定了基础。     

人乳头瘤病毒l6型E6-E7融合蛋白真核表达载体的构建与鉴定

目的构建人乳头瘤病毒l6型(HPV16)E6-E7融合蛋白真核表达载体,为研究其基因疫苗免疫活性奠定实验基础。方法 PCR扩增HPV16 E6-E7基因片段,将其连接到真核表达载体pcDNA3.1(+),构建真核表达载体pcDNA3.1(+)/HPV16 E6-E7,双酶切及测序鉴定。将质粒转染HeLa细胞,RT-PCR鉴定E6-E7基因在HeLa细胞中的表达。提取质粒免疫小鼠,利用免疫组化方法检测在其肌肉组织中的表达。结果成功构建了真核表达载体pcDNA3.1(+)/HPV16 E6-E7;在转染pcDNA3.1(+)/HPV16 E6-E7的细胞中检测到HPV16 E6-E7基因。在免疫该质粒的小鼠肌肉组织中可以检测到该质粒的蛋白表达。结论成功的构建的了真核表达载体pcDNA3.1(+)/HPV16 E6-E7,该载体能在HeLa细胞内以及小鼠骨骼肌细胞内有效表达。     

小鼠Oct4基因真核表达载体的构建及表达鉴定

目的:构建小鼠Oct4基因真核表达载体并对其表达进行鉴定.方法:以小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)cDNA为模板克隆Oct4基因编码区序列,将其通过定向克隆插入pcDNA3.1(+)栽体多克隆酶切位点(multiple cloning sites,MCS)中,形成pcDNA3.1(+)/Oct4重组载体.将pcDNA3.1(+)空载体和pcDNA3.1(+)/Oct4重组载体分别转染293T细胞,通过Western Blotting检测Oct4蛋白的表达.结果:成功检测到Oct4蛋白的表达.结论:成功构建了小鼠Oct4基因真核表达载体.     

小鼠Uncv基因克隆及真核表达

目的 克隆小鼠的Uncv基因并在真核细胞表达.方法 采用RT-PCR方法扩增小鼠皮肤组织中Uncv基因编码区,以真核表达质粒pcDNA 3.1-Flag为载体,构建Uncv真核表达质粒,将重组载体转染Hela细胞并用Western blot法检测基因表达.结果 构建Uncv基因真核表达载体pcDNA 3.1-Flag/Unev,重组质粒在Hela细胞中有效表达约95×103的融合蛋白.结论 成功构建真核表达载体pcDNA 3.1-Flag/Uncv,并且在真核细胞中有效表达,为研究Uncv基因生物学功能奠定基础.     

HBV PreS2+S/IFN-α融合基因真核表达载体的构建及其表达

构建含HBV PrdS2+S和IFN-α融合基因的真核表达载体pcDNA3.1.S2S/IFN-α并在真核细胞中进行表达.应用重叠延伸剪切技术(splicing by overlapping extension,简称SOE)经两次PCR获得嵌合基因片段S2S/IFN-α,回收后直接克隆到pcDNA3.1 V5/His TOPO TA克隆载体,得到真核重组载体pcDNA3.1.S2S/IFN-α.然后用脂质体法转染Vero E6细胞.对重组载体进行了限制性酶切及PCR鉴定,证明连接正确;经间接免疫荧光检测证实该重组载体能在真核细胞中表达插入的外源性基因编码的融合蛋白.真核表达载体pcDNA3.1.S2S/IFN-α的成功构建及在Vero E6细胞中的有效表达,为进一步探讨HBV感染的特异性免疫治疗提供了实验依据.     

HBV PreS2 S/IFN-α融合基因真核表达载体的构建及其表达

构建含HBVPrdS2 S和IFN α融合基因的真核表达载体pcDNA3.1.S2S/IFN α并在真核细胞中进行表达。应用重叠延伸剪切技术 (splicingbyoverlappingextension ,简称SOE)经两次PCR获得嵌合基因片段S2S/IFN α ,回收后直接克隆到 pcDNA3.1V5 /HisTOPOTA克隆载体 ,得到真核重组载体pcDNA3.1.S2S/IFN α。然后用脂质体法转染VeroE6细胞。对重组载体进行了限制性酶切及PCR鉴定 ,证明连接正确 ;经间接免疫荧光检测证实该重组载体能在真核细胞中表达插入的外源性基因编码的融合蛋白。真核表达载体pcDNA3.1.S2S/IFN α的成功构建及在VeroE6细胞中的有效表达 ,为进一步探讨HBV感染的特异性免疫治疗提供了实验依据     

hAng-1真核表达载体的构建及其在MSCs中的表达

目的:通过基因重组技术,构建人血管生成素-1(human angiopoietin 1,hAng-1)真核表达载体体系,并将其转染至大鼠的骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)内进行培养,进而验证hAng-1的表达.方法:将hAng-1编码序列(互补脱氧核糖核酸)通过酶切,插入至pcDNA3.1(+)质粒的多克隆位点,构建质粒pcDNA 3.1 (+)/hAng-1真核表达质粒;重组质粒经脂质体介导转染鼠MSCs.应用逆转录聚合酶联反应(RT-PCR)、蛋白免疫印迹法(Western blot)等方法检测hAng-1的表达情况.结果:pcDNA 3.1 (+)/hAng-1真核表达质粒转染鼠MSCs后,应用流式细胞仪检测,转染效率约为15％.同时应用RT-PCR能够检测出目的基因mRNA,Western blot能够检出hAng-1的蛋白表达.结论:本实验通过基因重组技术,构建的pcDNA3.1 (+)/hAng-1真核表达载体能够在转染的鼠MSCs中表达,且表达较为持续,为hAng-1基因应用于基因治疗的研究奠定了基础.     

小鼠EVL 基因的克隆和真核表达载体的构建

目的:构建小鼠EVL(Ena/VASP like)基因的真核表达载体,为深入研究EVL的功能奠定基础.方法:采用PCR方法,从小鼠cDNA文库中,扩增出1245bp的EVL编码区片段,经电泳、胶回收后连接入pMD- 18T载体中,测序鉴定正确.用BamHI和HincⅡ双酶切,定向克隆EVL编码区片段到真核表达载体pcDNA3.1中,用限制性内切酶酶切鉴定重组质粒正确后.将重组质粒转染入HELA细胞中,以RT-PCR检测EVL的mRNA的表达,以Western Blot检测EVL蛋白的表达.结果:酶切鉴定结果显示小鼠EVL编码区基因被成功克隆入真核表达载体pcDNA3.1中;RT-PCR和Western Blot结果以及免疫荧光染色显示Hela细胞中有EVL的mRNA和蛋白的表达.结论:成功获得pcDNA3.1 -EVL的真核表达载体,为进一步深入研究EVL蛋白的功能奠定了基础.     

人类VIM基因的克隆和真核表达载体的构建

目的:构建VIM(Vimentin)基因的真核表达载体,以深入研究VIM的功能及其在相关疾病中的作用.方法:从人cDNA文库中,以RT-PCR方法扩增出1401bp的VIM编码区片段,胶回收后连接入T.载体,测序鉴定.再用Hind Ⅲ和BamHI双酶切,将VIM编码区片段定向克隆到真核表达载体pcDNA3.1中,酶切鉴定重组质粒.将重组质粒转染NIH3T3细胞,分别以RT-PCR和Western Blot方法检测VIM的mRNA表达和蛋白表达.结果:将人VIM编码区基因成功克隆到真核表达载体pcDNA3.1中;继而转染NIH3T3细胞后,RT-PCR和Western Blot结果显示细胞可以表达VIM的mRNA和蛋白.结论:成功构建pcDNA3.1-VIM的真核表达载体,为进一步研究VIM基因的功能以及其在相关疾病中的作用奠定了基础.     

幽门螺杆菌Lpp20-IL2核酸疫苗免疫活性

【目的】观察pcDNA3.1(+)/Lpp20-IL2免疫C57BL/6小鼠后所产生的体液免疫和细胞免疫应答水平,为研制高效、新型的幽门螺杆菌核酸疫苗提供实验依据。【方法】构建pcDNA3.1(+)/Lpp20-IL2重组载体,并转染HeLa细胞,用Western-blot观察鉴定其在真核细胞得到表达后免疫C57BL/6小鼠,ELISA间接法测定小鼠血清中抗Lpp20IgG抗体水平,ELISA双抗体夹心法检测脾淋巴细胞培养上清中IFN-γ、IL4水平,MTT比色法检测脾淋巴细胞增殖反应,免疫荧光组化法检测Lpp20蛋白在小鼠肌肉组织中的表达情况。【结果】成功构建了pcDNA3.1(+)/Lpp20-IL2真核表达载体,且重组质粒能在HeLa细胞内有效表达目的蛋白;小鼠接种pcDNA3.1(+)/Lpp20-IL2核酸疫苗后能产生特异性IgG抗体,8w后ELISA测定血清抗体A450值明显升高。核酸疫苗pcDNA3.1(+)/Lpp20-IL2免疫组小鼠脾淋巴细胞经特异性抗原刺激后,培养上清中IFN-γ、IL4含量明显升高。pcDNA3.1(+)/Lpp20-IL2和pcDNA3.1(+)/Lpp20核酸疫苗组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数明显高于空质粒组和PBS组。Lpp20蛋白在小鼠肌肉组织中能够有效表达。【结论】幽门螺杆菌Lpp20-IL2融合基因核酸疫苗和Lpp20单基因核酸疫苗均能刺激机体产生较强细胞免疫应答和体液免疫应答,且前者能诱导更强的细胞免疫应答。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Comparison of BMI and percentage of body fat of Indian and German children and adolescents

Today, serious health problems as overweight and obesity are not just constricted to the developed world, but also increase in the developing countries (Prentice 2006, Ramachandram et al. 2002). Focusing on this issue, BMI and percentage of body fat were compared in 2094 schoolchildren from two cross-sectional studies from India and Germany investigated in 2008 and 2009. The German children are in all age groups significantly taller, whereas the Indian children show higher values in BMI (e.g. 12 years: Indian: around 22 kg/m2; German: around 19 kg/m2) and in the percentage of body fat (e.g. 12 years: Indian: around 27%; German: around 18-20%) in most of the investigated age groups. The Indian children have significantly higher BMI between 10 and 13 (boys) respectively 14 years (girls). Indian children showed significant higher percentage of body fat between 10 and 15 years (boys) and between 8 and 16 years (girls). The difference in overweight between Indian and German children was strongest at 11 (boys) and 12 (girls) years: 70% of the Indian but 20% of the German children were classified as overweight. In countries such as India that undergo nutritional transition, a rapid increase in obesity and overweight is observed. In contrast to the industrialized countries, the risk of overweight in developing countries is associated with high socioeconomic status. Other reasons of the rapid increase of overweight in the developing countries caused by different environmental or genetic factors are discussed.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission

The molecular characterisation of species and genotypes of Cryptosporidium and Giardia is essential for accurately identifying organisms and assessing zoonotic transmission. Results of recent molecular epidemiological studies strongly suggest that zoonotic transmission plays an important role in cryptosporidiosis epidemiology. In such cases the most prevalent zoonotic species is Cryptosporidium parvum. Genotyping and subtyping data suggest that zoonotic transmission is not as prevalent in the epidemiology of giardiasis. Molecular characterisation of Cryptosporidium and Giardia is a relatively recent application that is evolving as new genes are found that increase the accuracy of identification while discovering a greater diversity of species and yet unnamed taxa within these two important genera. As molecular data accumulate, our understanding of the role of zoonotic transmission in epidemiology and clinical manifestations is becoming clearer.     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.