表达PRRSV M蛋白重组腺病毒的构建及其免疫特性研究

本研究通过RT-PCR方法扩增猪繁殖与呼吸综合征病毒(PRRSV)S1株的M蛋白基因,将其克隆重组到人 血清5型腺病毒载体中,转染293细胞,制备重组腺病毒rAd-M。RT-PCR和IFA方法鉴定,结果表明rAd-M可表 达M基因的mRNA和M蛋白。纯化的rAd-M重组腺病毒经293细胞连续传25代,滴度稳定为107.8 TCID50／ mL。动物免疫试验结果表明,该重组腺病毒rAd-M能够刺激机体产生PRRSV的特异性抗体免疫和细胞免疫应 答反应,从而为PRRSV结构蛋白功能及其基因工程疫苗研究奠定了基础。     

表达猪繁殖与呼吸综合征病毒GP5和M融合蛋白的重组腺病毒的构建及其对小鼠的免疫原性

利用口蹄疫病毒(FMDV)2A蛋白具有自我裂解的功能,将其作为连接肽将猪繁殖与呼吸综合征病毒(PRRSV)的GP5和M蛋白编码基因串联,置于复制缺陷型腺病毒载体的表达盒中,通过一次转录和翻译,可以同时实现2个蛋白的表达,以发挥GP5蛋白的病毒中和优势和M蛋白的细胞免疫优势作用。分别利用RT-PCR、间接免疫荧光和Western blotting等方法,对获得的重组腺病毒(rAd-GP5-2A-M)进行检测,结果均证明GP5-2A-M蛋白不仅获得了正确表达,而且能自我裂解为GP5和M蛋白。以单独表达GP5(rAd-GP5)、M(rAd-M)和融合表达GP5-M(rAd-GP5-M)的重组腺病毒为对照,研究该重组腺病毒在小鼠体内诱导免疫应答情况,结果表明,虽然4种重组腺病毒均能诱导小鼠产生特异性抗体和细胞免疫反应,但重组腺病毒rAd-GP5-2A-M所诱导产生的体液免疫和细胞免疫应答水平最高。本研究结果提示,利用具有自动裂解功能的FMDV2A多肽构建PRRSV复合基因工程疫苗是一种切实可行的新策略,也为构建其他动物病毒病的基因工程疫苗提供了新思路。     

人呼吸道合胞病毒M蛋白非复制型重组腺病毒的构建和鉴定

人呼吸道合胞病毒（human respiratory syncytial virus, RSV)基质蛋白（matrix protein,M）在RSV形态发生上具有重要作用,因含有CTL抗原表位,在疫苗研究上具有一定意义。为此,应用RT-PCR 方法从感染RSV的HEp-2 细胞中扩增获得M蛋白基因,构建了含M基因的非复制型重组腺病毒并进行表达和鉴定。基因序列分析显示RSV M基因仅有一处碱基发生错义突变。非复制型重组腺病毒DNA分子FGAd/RSVM转染293细胞,观察到细胞出现CPE,RT-PCR发现M基因有转录,Western blotting及间接免疫荧光分析检测到M蛋白。成功克隆A亚型RSV Long株M基因,并获得一株可表达A亚型RSV M蛋白的非复制型重组腺病毒FGAd/RSVM,可用于体内研究观察其免疫效果及免疫保护作用。     

猪α干扰素重组腺病毒的构建及其抗口蹄疫病毒活性研究

本研究利用RT-PCR方法扩增猪α干扰素成熟蛋白基因后构建了重组腺病毒质粒pAd-poIFN-a,经PacI酶切后转染HEK-293A细胞,3次噬斑纯化后获得了重组腺病毒rAd-poIFN-α.该重组腺病毒于HEK-293A细胞连续传代至20代滴度稳定,为107 TCID50/mL.RT-PCR检测证明目的基因在mRNA水平上可有效表达;在PK-15细胞上可以检测到较强的抗猪口蹄疫病毒活性,从而为研究猪口蹄疫免疫防治新技术奠定了重要基础.     

猪繁殖与呼吸综合征病毒GP5蛋白重组腺病毒的构建与免疫原性测定

应用RT-PCR方法扩增出猪繁殖与呼吸综合征病毒国内分离株S1毒株的GP5基因序列,然后通过KpnI和XhoI酶切位点把该基因克隆入经过同样双酶切的穿梭载体pShuttle-CMV中.重组穿梭载体经过酶切和PCR鉴定后进行测序,证明所克隆入的基因以正确的阅读框插入.获得的重组穿梭载体经线性化后与腺病毒骨架载体共转化大肠杆菌BJ5183菌株.纯化的重组腺病毒质粒经酶切线性化后转染293细胞获得重组腺病毒.重组腺病毒经纯化后进行RT-PCR和间接免疫荧光鉴定,证明了用PRRSVGP5蛋白基因所构建的重组腺病毒成功的表达了GP5蛋白.猪体免疫试验后收集血清进行中和实验证明所构建的重组腺病毒在猪体内能够诱导产生中和抗体,因此我们所构建的重组腺病毒可以作为PRRSV基因工程疫苗研究候选病毒株.     

猪α干扰素重组腺病毒的构建及其抗口蹄疫病毒活性研究

本研究利用RT-PCR方法扩增猪α干扰素成熟蛋白基因后构建了重组腺病毒质粒pAd-poIFN-α,经PacI酶切后转染HEK-293A细胞,3次噬斑纯化后获得了重组腺病毒rAd-poIFN-α。该重组腺病毒于HEK-293A细胞连续传代至20代滴度稳定,为107TCID50/mL。RT-PCR检测证明目的基因在mRNA水平上可有效表达;在PK-15细胞上可以检测到较强的抗猪口蹄疫病毒活性,从而为研究猪口蹄疫免疫防治新技术奠定了重要基础。     

猪繁殖与呼吸综合征病毒GP5蛋白重组腺病毒的构建与免疫原性测定

应用RT-PCR方法扩增出猪繁殖与呼吸综合征病毒国内分离株S1毒株的GP5基因序列,然后通过KpnI和XhoI酶切位点把该基因克隆入经过同样双酶切的穿梭载体pShuttle-CMV中。重组穿梭载体经过酶切和PCR鉴定后进行测序,证明所克隆入的基因以正确的阅读框插入。获得的重组穿梭载体经线性化后与腺病毒骨架载体共转化大肠杆菌BJ5183菌株。纯化的重组腺病毒质粒经酶切线性化后转染293细胞获得重组腺病毒。重组腺病毒经纯化后进行RT-PCR和间接免疫荧光鉴定,证明了用PRRSVGP5蛋白基因所构建的重组腺病毒成功的表达了GP5蛋白。猪体免疫试验后收集血清进行中和实验证明所构建的重组腺病毒在猪体内能够诱导产生中和抗体,因此我们所构建的重组腺病毒可以作为PRRSV基因工程疫苗研究候选病毒株。     

表达流感病毒神经氨酸酶基因的重组腺病毒的构建

通过RT-PCR方法扩增流感病毒神经氨酸酶基因,将其克隆到腺病毒穿梭载体pTrackCMV,此重组质粒与腺病毒DNA共转化大肠杆菌BJ5183,通过细菌内同源重组获得重组腺病毒DNA,将其转染293细胞获得重组腺病毒.经PCR证实目的基因已整合至腺病毒基因组中,western blot检测到神经氨酸酶的表达.重组病毒经滴鼻和灌胃两种途径免疫小鼠,结果表明2次免疫后滴鼻组和灌胃组均产生明显的免疫应答反应,滴鼻组的免疫效果优于灌胃组.     

携带PTEN基因的重组腺病毒表达载体构建的研究

构建携带抑癌基因PTEN(Phosphatase and temin homolog deleted on chromosome ten)的重组腺病毒表达裁体,为研究PTEN的功能和作用机制奠定基础.采用RT-PCR法从大鼠海马神经元扩增目的基因PTEN,克隆人含绿色荧光蛋白(Green fluorescence protein),GFP基因的pAdTrack-CMV穿梭质粒,在含有腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌内进行同源重组;获得重组腺病毒质粒,经Pacl线性化后,转染AD293细胞.结果表明,感染腺病毒载体的AD293细胞表达GFP基因,随着时间逐渐增强,并且出现明显的细胞病变效应(Cytopathic effect,CPE),经PCR对传代的Ad-PTEN分析证实得到目的基因.成功构建了携带PTEN基因的腺病毒表达载体,为研究PTEN的功能和作用机制奠定了基础.     

腹腔注射重组腺病毒诱导的免疫反应

为研究重组腺病毒接种实验动物后的免疫反应性,利用RT-PCR方法,从HAV的RNA中克隆了结构蛋白基因插入穿梭质粒pXCX2Not I,通过磷酸钙-DNA共沉淀技术,将复制缺陷型腺病毒载体与线形化的pXCX2 CMV-HAV共转染293细胞.一系列检测方法证明产生了重组腺病毒rAdHAV.纯化后的rAdHAV滴度为1×109TCID50/mL,腹腔注射免疫昆明种小白鼠后,可诱导产生抗HAV IgG和HAV中和抗体.复制缺陷型腺病毒可作为发展基因工程病毒疫苗载体的有效系统.     

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、...     

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.     

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.     

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。     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000