重组铜绿假单胞菌外膜蛋白OprI的原核表达及多克隆抗体制备

[目的]构建铜绿假单胞菌外膜蛋白OprI原核载体,表达、纯化OprI蛋白,制备并鉴定小鼠多克隆抗体。[方法]通过分子克隆获得OprI蛋白的表达菌株;利用SDS-PAGE电泳切胶纯化、尿素梯度复性获得OprI蛋白,免疫小鼠制备OprI蛋白多克隆抗体;采用ELISA法检测抗体滴度;Western blotting检测抗血清特异性;DNAMAN与MEGA软件分析OprI蛋白系统进化关系。[结果]OprI重组载体双酶切、DNA测序鉴定结果,以及蛋白表达、纯化条带大小与预测一致。ELISA法获得OprI抗血清滴度达1:3200倍,Western blotting证实抗血清具有很好的特异性。OprI序列的系统发生分析发现不同细菌存在很高的同源性。[结论]成功克隆、表达与纯化OprI蛋白,制备并鉴定小鼠多克隆抗体。为OprI蛋白免疫学功能研究奠定基础。     

铜绿假单胞菌外膜蛋白Ⅰ的原核表达、纯化及免疫保护作用研究

铜绿假单胞菌外膜蛋白Opr I具有较强的免疫原性,在疫苗上具有开发前景。利用分子方法获得Opr I蛋白的表达菌株。Western blotting验证表明抗体能与Opr I蛋白特异性结合。SDS-PAGE电泳切胶纯化获得Opr I蛋白。将Opr I蛋白免疫小鼠并攻毒铜绿假单胞菌,发现Opr I蛋白激活的特异性免疫对小鼠铜绿假单胞菌感染的保护率达到57.14%,与对照组相比较达到显著性。采用正交试验设计,获得Opr I菌株最佳诱导表达条件为:加IPTG菌夜OD600值0.8,加IPTG终浓度0.3 mmol/L,诱导时间8 h,诱导温度28℃;菌株最佳培养条件为转速230 r/min,葡萄糖浓度0%,装液量50 m L。     

重组人骨硬化蛋白的表达、纯化及多克隆抗体制备

[目的]构建人骨硬化蛋白(Sclerostin,SOST)原核载体,表达、纯化SOST蛋白,制备与鉴定小鼠多克隆抗体。[方法]通过分子克隆获得SOST蛋白的表达菌株;利用SDS-PAGE电泳切胶纯化、尿素梯度复性获得SOST蛋白,免疫小鼠制备SOST蛋白多克隆抗体;采用ELISA法检测抗体滴度;Western blotting检测抗血清特异性;DNAMAN与MEGA软件分析SOST蛋白系统进化关系。[结果]SOST重组载体双酶切、DNA测序鉴定结果,以及蛋白表达、纯化条带大小与预测一致。ELISA法获得SOST抗血清滴度达1:6 400倍,Western blotting证实抗血清具有很好的特异性。SOST序列的系统发生分析发现动物具有显著不同的进化趋势。[结论]成功克隆、表达与纯化SOST蛋白,制备并鉴定小鼠多克隆抗体。为SOST蛋白功能研究奠定基础。     

鸭疫里默氏杆菌外膜蛋白生物学特性研究

血清2型鸭疫里默氏杆菌强毒菌株体外传200代获得了无毒力无免疫原性菌株,采用超声波裂解和超速离心法提取二株菌的外膜蛋白, 以比较分析鸭疫里默氏杆菌外膜蛋白的生物学特性。电镜观察细菌超微结构显示传代菌株外膜膜密度降低, 外膜泡的数量明显减少, 细胞质不均匀、内有空泡产生;免疫印迹结果表明二株菌的外膜蛋白免疫原性多肽存在明显区别;原代菌株的外膜蛋白仅与2型RA抗体出现特异性凝集, 而传代菌株的外膜蛋白与 1、2、10与11型RA抗体均出现凝集;二株菌的外膜蛋白均可诱导雏鸭产生抗体, 但原代菌株外膜蛋白诱导雏鸭产生抗体滴度显著高于200代次菌株;原代菌株外膜蛋白免疫鸭对同源RA菌株的攻击可产生100%的免疫保护, 而传代菌株外膜蛋白免疫鸭对同源RA菌株的攻击不产生免疫保护。序列分析显示两者的外膜蛋白A同源性达到99.9%。结果表明强毒菌株的外膜蛋白为良好的亚单位疫苗候选, 体外连续传代对RA外膜蛋白生物学特性影响显著。     

鸭疫里默氏杆菌外膜蛋白生物学特性研究

血清2型鸭疫里默氏杆菌强毒菌株体外传200代获得了无毒力无免疫原性菌株,采用超声波裂解和超速离心法提取二株菌的外膜蛋白, 以比较分析鸭疫里默氏杆菌外膜蛋白的生物学特性。电镜观察细菌超微结构显示传代菌株外膜膜密度降低, 外膜泡的数量明显减少, 细胞质不均匀、内有空泡产生;免疫印迹结果表明二株菌的外膜蛋白免疫原性多肽存在明显区别;原代菌株的外膜蛋白仅与2型RA抗体出现特异性凝集, 而传代菌株的外膜蛋白与 1、2、10与11型RA抗体均出现凝集;二株菌的外膜蛋白均可诱导雏鸭产生抗体, 但原代菌株外膜蛋白诱导雏鸭产生抗体滴度显著高于200代次菌株;原代菌株外膜蛋白免疫鸭对同源RA菌株的攻击可产生100%的免疫保护, 而传代菌株外膜蛋白免疫鸭对同源RA菌株的攻击不产生免疫保护。序列分析显示两者的外膜蛋白A同源性达到99.9%。结果表明强毒菌株的外膜蛋白为良好的亚单位疫苗候选, 体外连续传代对RA外膜蛋白生物学特性影响显著。     

弧菌外膜蛋白OmpU的免疫交叉反应性和交叉保护性北大核心CSCD

【目的】通过对弧菌外膜蛋白Omp U的克隆、表达以及免疫学特性分析,明确外膜蛋白Omp U是否为弧菌的共同抗原,并具有免疫交叉反应性和交叉保护性。【方法】对弧菌外膜蛋白omp U基因进行克隆和生物信息学分析。分别制备副溶血弧菌、溶藻弧菌、创伤弧菌、拟态弧菌和霍乱弧菌的Omp U重组蛋白抗血清,对Omp U的免疫交叉反应特性以及抗原表位定位情况进行比较分析。以霍乱弧菌的Omp U重组蛋白免疫小鼠后,再以多种弧菌进行攻毒,分析其交叉免疫保护作用。【结果】外膜蛋白Omp U在弧菌种内和种间相似性分别为73.0%–100%和58.6%–89.0%,并至少存在9个保守的B细胞抗原表位。Omp U重组蛋白抗血清在弧菌种内和种间均产生显著的免疫交叉反应,识别弧菌中分子量35–40 k Da的同源蛋白。副溶血弧菌ATCC17802、创伤弧菌ATCC27562和拟态弧菌ATCC33653来源的Omp U重组蛋白抗体能识别供试菌株,提示这些菌株的Omp U抗原表位定位于细胞表面。Omp U重组蛋白对免疫后的小鼠具有交叉免疫保护作用,攻毒实验后小鼠相对存活率(RPS)为43.0%–100%。【结论】上述结果表明,外膜蛋白Omp U是弧菌中一种保守的共同抗原,具有免疫交叉保护性,可以作为弧菌广谱疫苗的候选抗原。     

利用免疫蛋白质组学方法筛选高效的疫苗

厦门大学利用免疫蛋白质组学发展了一种快速筛选高效候选疫苗的方法。这种方法分为三个步骤：首先用免疫蛋白质组学方法筛选外膜蛋白的主要免疫原;接着,用毒性细菌在接种后激发以从免疫原中找出能够诱导中和性抗体产生的蛋白;最后,通过评估中和能力来筛选候选疫苗。通过质谱分析能够获得候选者的遗传信息以进行进一步的基因克隆。     

应用人型单克隆抗体治疗感染疾病

<正> 接受免疫仰制剂治疗癌症、重度烧伤等免疫功能低下的患者,常招致严重的绿脓杆菌(P、aeruginosa)感染。此外,可对现有抗生素很快产生抗药性,用化学疗法很难治疗。 Millican等(1958年)首次报道抗绿脓杆菌免疫血清抗体疗法有实用价值。以后,许多作者对抗绿脓杆菌各种成分的抗体进行了深入研究,结果发现脂多糖(LPS,存在于菌体外膜)抗体,可借助吞噬细胞的免疫吞噬作用而感染。用鼠型单克隆抗体(McAp)试验也有同样的结果,并发现抗外膜蛋白McAp的防御感染作用比抗LPSMcAp效果差。 最近,制备抗绿脓杆菌脂多糖的人型McAp已获成功,产生人型McAp的细胞是采用Epstein-Barr病毒(EBV)转化法或杂交细胞瘤法。作者对健康人未梢血淋巴细胞的病毒转化细胞产生的抗绿脓杆菌人型     

棉蚜His-CYP6J1融合蛋白的分离纯化及多克隆抗体的制备

通过亲和层析法纯化棉蚜His-CYP6J1融合蛋白,制备及鉴定其多克隆抗体。采用镍离子金属螯合亲和层析柱分离纯化棉蚜His-CYP6J1融合蛋白,SDS-PAGE检测纯化产物。用纯化得到的His-CYP6J1融合蛋白免疫小鼠,制备抗棉蚜P450CYP6J1多克隆抗体;采用ELISA法检测抗体效价;免疫组化法检测抗血清特异性。结果表明,纯化的His-CYP6J1融合蛋白免疫小鼠后得到多抗血清,ELISA法检测抗血清效价达1∶200 000。免疫组化结果显示,此多克隆抗体能够与棉蚜P450 CYP6J1天然蛋白特异性结合,却在棉铃虫没有发现相应的结合反应。上述结果对研究棉蚜单一P450蛋白结构、功能及其在棉蚜抗药性形成过程的作用奠定基础。     

钼还原菌中TrxR的原核表达及多克隆抗体制备

[目的]构建枯草芽孢杆菌硫氧还蛋白还原酶(thioredoxin reductase,TrxR)原核载体,表达、纯化TrxR重组蛋白,制备并鉴定多克隆抗体。[方法]通过分子克隆获得TrxR蛋白的表达菌株;利用镍离子亲和层析获得纯化的TrxR重组蛋白,免疫兔子制备TrxR蛋白多克隆抗体;采用ELISA法测定抗体效价;Western Blot检测抗血清的特异性。[结果]TrxR重组载体双酶切结果与DNA测序鉴定结果一致,蛋白表达纯化条带大小与预测一致。ELISA法测定抗血清效价为7×104,Western Blot证实抗血清有较高的特异性。[结论]成功克隆、表达与纯化TrxR重组蛋白,制备并鉴定兔子多克隆抗体,为TrxR的生物学功能研究奠定基础。     

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