肽基脯氨酰异构酶在细胞核内的功能

有许多酶具有调节转录因子的功能,例如蛋白激酶、磷酸酯酶和乙酰转移酶等。目前Ｌｅｖｅｒｓｏｎ等提出一种新的酶调节机制：肽基脯氨酰异构酶（Ｐｅｐｔｉｄｙｌ－ｐｒｏｌｙｌｉｓｏｍｅｒａｓｅｓ,ＰＰＩａｓｅ）参与调节转录因子的活性［１］。转录因子ｃ－Ｍｙｂ结...     

脑膜炎奈瑟氏菌外膜蛋白NhhA的克隆表达及免疫学分析

通过克隆脑膜炎奈瑟氏菌NhhA基因GNA0992于原核表达载体pET 20b,在大肠杆菌BL21（DE3）中实现了可溶性表达,表达量约占菌体蛋白总量的20%。经初步纯化后免疫小鼠,对其免疫原性进行了初步分析。结果显示,经3次腹腔免疫,血清IgG滴度达到23186,同时杀菌力实验显示NhhA能诱导针对B群脑膜炎奈瑟氏菌的补体依赖的杀菌反应。证明了NhhA是一种良好的抗原,为疫苗开发的蛋白靶标筛选工作奠定了基础。     

意大利蜜蜂丙糖磷酸异构酶基因的克隆及其原核表达与纯化

从意大利蜜蜂Apis mellifera ligustica的肌肉组织中提取总RNA,采用RT-PCR的方法克隆蜜蜂第16号染色体上的丙糖磷酸异构酶基因的cDNA序列,将测序结果(GenBank登录号EU76098)与推导的氨基酸序列分别与GenBank中的其他物种进行同源比对分析。结果表明,该基因全长744bp,为完整的阅读框,编码247个氨基酸,成熟蛋白的理论分子量为26.89kD。比对结果显示AmTPI与家蚕、德国小镰、黄粉虫、丽蝇蛹集金小蜂、水稻等物种的基因相似性达69%以上,蛋白相似性达59%以上。将目的基因克隆到pGEX-4T-2融合表达载体上,并在大肠杆菌中得到成功表达,4h的表达量为总蛋白的42.1%。为了进一步探讨产物的酶学特性,实验还对表达产物进行纯化与浓缩。实验还构建增强型荧光真核表达质粒,为进一步研究AmTPI在真核细胞中的表达情况奠定基础。     

CD36原核表达载体的构建及生物信息学分析

[目的]对CD36基因进行体外克隆表达,建立CD36基因cDNA在大肠杆菌中表达的实验技术以及进行CD36蛋白三级结构的预测并利用生物信息学方法进行分析。[方法]以人血液RNA为模板,RT-PCR扩增CD36的基因序列;并构建原核表达质粒pET-32a-CD36转化到大肠杆菌DE3中后,经过IPTG诱导表达后进行SDS-PAGE验证。[结果]成功克隆了人CD36基因并构建出原核表达质粒,成功转入表达菌大肠杆菌DE3中后经IPTG诱导成功表达,且在1 mmol/L的诱导剂浓度下培养6 h表达量最佳。利用Phyre2和Abcpred软件预测蛋白质结构和B细胞抗原表位,B细胞抗原表位评分大于0.85的CD36的B细胞抗原表位有8个。[结论]成功建立了人CD36基因的原核表达的实验技术,并对CD36的结构及B细胞抗原表位进行了预测,为后续研究CD36基因的功能和抗体制备提供方向和奠定基础。     

肠炎沙门菌肽脯氨酰顺反异构酶SlyD的基因敲除、表达及酶学特征

【目的】以肠炎沙门菌肽脯氨酰顺反异构酶SlyD为对象,构建基因缺失株及表达纯化该蛋白,为研究其在肠炎沙门菌致病性与应激等方面的作用奠定基础。【方法】参考Gen Bank登录的肠炎沙门菌基因组序列设计用于slyD基因敲除及原核表达的特异引物,运用自杀质粒介导的同源重组技术对肠炎沙门菌C50041 slyD基因进行敲除,构建C50041ΔslyD缺失株;原核表达SlyD蛋白,通过α-糜蛋白酶耦联法对其PPIase活性进行测定;利用生物信息学相关软件,分析SlyD蛋白的氨基酸序列及功能域。【结果】PCR鉴定与测序结果证明成功构建了肠炎沙门菌C50041ΔslyD缺失株,其生长特性与野生株基本一致;SDS-PAGE及PPIase活性分析表明,获得了具有生物活性的可溶性SlyD蛋白;生物信息学分析显示SlyD蛋白由FKBP样肽脯氨酰顺反异构酶结构域、分子伴侣功能域和金属结合区域3个功能区域组成。【结论】成功获得了肠炎沙门菌C50041ΔslyD缺失株和具有PPIase活性的重组SlyD蛋白。     

水貂阿留申病毒VP2蛋白主要抗原表位基因原核表达及其检测应用

将构建好的重组质粒pMD-VP2a、pMD-VP2b分别经双酶切获得基因片段VP2a、VP2b,分别将其定向插入到原核表达载体pMAL-c2中,构建原核表达载体pMAL-VPa和pMAL-VPb。阳性重组质粒转化宿主菌TB1,经IPTG诱导,两段基因均获得表达;Westernblot分析表明表达蛋白具有良好的抗原性。用KCI染色后,切胶回收的方法对表达蛋白进行纯化,以纯化的目的蛋白作为诊断抗原初步建立了水貂阿留申的VP2-CIEP诊断方法。结果表明该方法具有较高的灵敏性和特异性,与传统的CIEP方法的检出符合率达94.3%。     

淋病奈瑟菌感染合并尖锐湿疣患者光动力疗法治疗后皮损处朗格汉斯细胞的变化及其免疫机制研究

探讨淋病奈瑟菌（NG）感染合并尖锐湿疣（CA）患者光动力疗法（PDT）治疗后皮损处朗格汉斯细胞（LC）的变化及其免疫机制,为该类患者的治疗提供参考。

前瞻性纳入2020年3月至2021年3月我院收治的103例NG感染合并CA患者作为观察对象,通过随机数字表法分为观察组（n=53）和对照组（n=50）,并纳入5例同期来本院进行包皮环切术的男性作为正常组。对照组患者采用CO₂激光治疗,观察组患者采用PDT治疗,正常组无治疗。采用酶联免疫吸附法（ELISA）分别检测观察组、对照组患者治疗前后外周血中IL-2、IFN-γ水平,并比较两组患者临床疗效、复发率、不良反应发生率及21种人乳头瘤病毒（HPV）基因分布情况。采用免疫组化标记方法对观察组患者PDT治疗前后CA皮损组织和正常组中正常包皮组织进行染色,并于高倍光镜下观察LC的数量及形态变化,分析其免疫机制。

（1）观察组患者治疗总有效率显著高于对照组（98.11% vs 84.00%,P＜0.05）。（2）观察组患者疾病总复发率（7.55%）、不良反应总发生率（5.66%）均显著低于对照组（32.00%、40.00%）（均P＜0.05）。（3）治疗后,观察组患者IL-2、IFN-γ水平均显著高于对照组（均P＜0.05）。（4）103例NG感染合并CA患者中低危型HPV感染率为57.28（59/103）,高危型感染率为14.56%（15/103）,中危型感染率为5.83%（6/103）,混合感染率为22.33%（23/103）;同时观察组患者治疗后HPV清除率为83.02%,显著高于对照组的52.00%（P＜0.05）。（5）治疗前观察组患者LC数量较正常组显著偏少,胞体较正常组偏小,分布不规则,且多数LC结构不完整,不具备LC典型特征,部分皮损处表皮内无LC,但其真皮内可见到不典型的LC。（6）观察组患者皮损处治疗前LC数量较正常组显著偏少,治疗后LC数量即刻增加,治疗后7 d恢复至基线水平;同时正常组CD1a染色阳性细胞密度为6.48%,显著高于观察组患者治疗前后各时间点（均P＜0.05）。

PDT对NG感染合并CA患者的临床疗效显著,复发率及不良反应发生率较低,且可正向调节患者外周血IL-2、IFN-γ水平,参与机体特异性免疫应答,改变皮损组织中LC的数量和形态。

     

李斯特菌溶血素基因的原核表达及其生物学特性

李斯特菌溶血素(LLO)是产单核细胞李斯特菌的主要毒力因子,利用PCR技术从血清型4b的产单核细胞李斯特菌菌株中扩增出编码LLO的hly基因,经克隆筛选和测序鉴定后,构建成该基因的原核表达质粒pGEX6P1hly,SDSPAGE结果表明：LLO与谷胱甘肽在大肠杆菌中已融合表达,融合蛋白的分子量为82kD;溶血实验证明融合蛋白具有较强的裂解真核细胞膜的作用,表明表达产物LLO具有生物活性,其溶血效价达2.26×101.4 HU/mg,这为进一步研究其致病与免疫机理、单抗研制和疫苗设计提供了条件。     

猪瘟病毒NS2-3抗原集中区基因的原核表达及鉴定

为获得猪瘟病毒(classical swine fever virus, CSFV) NS2-3抗原集中区蛋白,并建立CSFV抗体快速检测方法.本研究以CSFV全长基因组质粒为模板,PCR扩增NS2-3抗原表位集中区,利用扩增片段和克隆载体,构建重组表达质粒,命名为pET32a-NS2-3-1.重组表达质粒转化Rosetta (DE3)细胞,利用IPTG诱导表达, SDS-PAGE电泳和Western-blot鉴定重组表达产物.结果表明,重组质粒pET32a-NS2-3-1在28℃诱导5 h得到高效表达,重组蛋白能够与兔抗CSFV阳性血清发生反应.获得CSFV NS2-3抗原集中区蛋白,并且获得的重组蛋白具有抗原性,能够作为CSFV抗体检测的抗原.     

沙门菌毒力基因spvB的原核表达及其多克隆抗体的制备

目的:构建沙门菌毒力基因spvB的原核表达载体,诱导表达纯化SpvB蛋白并以其为抗原免疫小鼠,制备多克隆抗体。方法:利用生物信息学软件对SpvB进行分析,选取抗原性较高、易表达的氨基酸序列作为克隆序列,以携带spvB基因的鼠伤寒沙门菌为模板,PCR扩增目的片段后与原核表达载体pET28a(+)连接;将质粒pET28a-SpvB转化大肠埃希菌BL21(DE3)后诱导表达并纯化。目的蛋白免疫小鼠,制备抗SpvB多克隆抗体,Western blot检测抗体特异性。结果:成功构建spvB原核表达载体,经IPTG诱导结果显示,重组蛋白表达且主要存在于包涵体中,将纯化后的蛋白免疫小鼠Western blot检测血清中抗体与SpvB特异性结合。结论:获得具有免疫原性的SpvB蛋白及其多克隆抗体,为进一步研究该基因的功能奠定基础。     

pUB307 mobilizes resistance plasmids from Escherichia coli into Neisseria gonorrhoeae

Summary The plasmid pUB307, a derivative of RP1, is a conjugative, broad-host-range plasmid. We have shown that this element mobilizes gonococcal resistance plasmids from Escherichia coli to Neisseria gonorrhoeae, thus providing evidence that extrachromosomal elements can efficiently enter gonococci by conjugation. Furthermore, pUB307 can also be used as a helper element to mobilize the cloning vector pLES2 into N. gonorrhoeae. This finding significantly increases the usefulness of pLES2 as a shuttle vector between E. coli and gonococcus.     

Use of a non-selective transformation technique to construct a multiply restriction/modification-deficient mutant ofNeisseria gonorrhoeae

A technique that allows for easy identification of transformants ofNeisseria gonorrhoeae in the absence of selective pressure has been developed. A suicide vector that contains a gonococcal DNA uptake sequence was constructed to aid in DNA uptake. In this transformation procedure, a limiting number of cells is incubated with an excess amount of DNA, and the mixture is plated onto a non-selective medium. At least 20% of the resulting colonies contained cells that had been transformed. This strategy was utilized to construct specific deletions of the S.NgoI, II, IV, V and VII restriction-modification (R/M) genes. All five deletions were successfully incorporated into the chromosome of FA19, producing strain JUG029. Strain JUG029 could be transformed with non-methylated plasmid DNA while strain FA19 could not be transformed with such DNA. The development of a simple, non-selective transformation technique, coupled with the construction of a strain that is more permissive for DNA-mediated transformation, will aid in genetic manipulations of the gonococcus.     

乳房链球菌GapC蛋白的表达及其B细胞抗原表位的预测与鉴定

乳房链球菌Streptococcus uberis的GapC蛋白是一种位于该菌表面的具有甘油醛-3-磷酸脱氢酶活性的蛋白,其参与细胞活动,表现出多种生物学活性,此外还具有良好的抗原性。文中旨在对乳房链球菌GapC蛋白可能的B细胞抗原表位进行预测,分析和验证候选表位肽的免疫原性。利用S. uberis分离株RF5-1克隆gapC基因,构建重组表达质粒pET-28a-GapC,诱导表达GapC重组蛋白,并以纯化蛋白免疫家兔,获得抗GapC多抗。利用生物信息学软件预测并分析GapCB细胞抗原表位的三维结构和空间位置及对GapC蛋白及表位的同源性比较。结果表明,表达纯化了44kDa的GapC蛋白具有良好的反应性。利用表位预测软件筛选并合成针对S.uberisGapC蛋白的6个线性和3个构象优势B细胞表位多肽,三维结构的分析显示,筛选的多肽具有良好的抗原表位形成条件。以纯化的S.uberis GapC蛋白免疫家兔制备多抗,通过间接ELISA对抗原表位进行鉴定。ELISA检测结果显示,9条抗原表位肽均可不同程度地与抗GapC多抗反应,其中表位266AANDSYGYTEDPIVSSD282与多抗反应...     

Characterization of a subunit structure and stability of the recombinant porin fromNeisseria gonorrhoeae

An outer membrane PIA protein fromNeisseria gonorrhoeae strain FA19 was expressed inEscherichia coli and refoldedin vitro in the presence of zwitterionic detergent. Its proper folding and subunit organization was confirmed by comparison with the native counterpart. The unfolding of PIA has been investigated using fluorescence spectroscopy and analytical size-exclusion chromatography methods. Analysis of the denaturation pathway of the PIA revealed that it forms an unusually labile quaternary structure. In the presence of 1 M guanidinium chloride (GdmCl) or upon heating up to 50°C, dissociation of the PIA oligomer was observed resulting in the formation of folded monomeric intermediates. Unfolding of monomers occurs at 80°C or in the presence of 4.3 M GdmCl, indicating high intrinsic stability toward both GdmCl and elevated temperatures. Both oligomeric and monomeric forms of PIA exhibited affinity to the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and bind withK _d=80 and 130 μM, respectively. Denaturation of the PIA completely abolished affinity to ANS, suggesting that hydrophobicity is a property of the folded state of the porin.     

Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12

Summary The genes xy1A and xy1B were cloned together with their promoter region from the chromosome of Klehsiella pneumoniae var. aerogenes 1033 and the DNA sequence (3225 bp) was determined. The gene xy1A encodes the enzyme xylose isomerase (XI or XylA) consisting of 440 amino acids (calculated M_r of 49 793). The gene xy1B encodes the enzyme xylulokinase (XK or Xy1B) with a calculated M, of 51 783 (483 amino acids). The two genes successfully complemented xy1 mutants of Escherichia coli K12, but no gene dosage effect was detected. E. coli wild-type cells which harbored plasmids with the intact xylA _Kp 5 upstream region in high copy number (but lacking an active xy1B gene on the plasmids) were phenotypically xylose-negative and xylose isomerase and xylulokinase activities were drastically diminished. Deletion of 5 upstream regions of xy1A on these plasmids and their substitution by a lac promoter resulted in a xylose-positive phenotype. This also resulted in overproduction of plasmid-encoded xylose isomerase and xylulokinase activities in recombinant E. coli cells.     

Structural and functional studies of FkpA from Escherichia coli, a cis/trans peptidyl-prolyl isomerase with chaperone activity

The protein FkpA from the periplasm of Escherichia coli exhibits both cis/trans peptidyl-prolyl isomerase (PPIase) and chaperone activities. The crystal structure of the protein has been determined in three different forms: as the full-length native molecule, as a truncated form lacking the last 21 residues, and as the same truncated form in complex with the immunosuppressant ligand, FK506. FkpA is a dimeric molecule in which the 245-residue subunit is divided into two domains. The N-terminal domain includes three helices that are interlaced with those of the other subunit to provide all inter-subunit contacts maintaining the dimeric species. The C-terminal domain, which belongs to the FK506-binding protein (FKBP) family, binds the FK506 ligand. The overall form of the dimer is V-shaped, and the different crystal structures reveal a flexibility in the relative orientation of the two C-terminal domains located at the extremities of the V. The deletion mutant FkpNL, comprising the N-terminal domain only, exists in solution as a mixture of monomeric and dimeric species, and exhibits chaperone activity. By contrast, a deletion mutant comprising the C-terminal domain only is monomeric, and although it shows PPIase activity, it is devoid of chaperone function. These results suggest that the chaperone and catalytic activities reside in the N and C-terminal domains, respectively. Accordingly, the observed mobility of the C-terminal domains of the dimeric molecule could effectively adapt these two independent folding functions of FkpA to polypeptide substrates.     

牛源致病性蜡样芽孢杆菌溶血素BL亚基的原核表达及其生物学活性分析

蜡样芽孢杆菌属于革兰氏阳性菌,分布广泛,具有一定的致病性。不同的蜡样芽孢杆菌携带有不同的毒力因子,这直接决定了蜡样芽孢杆菌株致病性的差异。研究和探讨毒力因子分布以及具体毒素的生物活性有助于对蜡样芽孢杆菌病采取更科学的防控。本研究通过原核表达系统将溶血素BL三亚基重组表达,并对表达蛋白进行了纯化和部分生物学活性的检测。研究结果表明,牛源致病性蜡样芽孢杆菌溶血素BL可以在原核表达系统中成功表达和纯化,牛源致病性蜡样芽孢杆菌溶血素BL拥有溶血性、细胞毒性、很好的免疫原性以及对小鼠具有一定的免疫保护性。本研究表达了溶血素BL三亚基,并探究了牛源致病性蜡样芽孢杆溶血素BL的生物学活性。本研究为进一步揭示蜡样芽孢杆菌溶血素BL的致病作用机制和建立针对牛源致病性蜡样芽孢杆菌病的检测方法奠定了理论基础。     

‘潘那利’番茄碱性螺旋环螺旋转录因子基因的克隆与表达分析

碱性螺旋环螺旋(basic/Helix Loop Helix, bHLH)转录因子是植物最大的转录因子家族之一,其广泛参与植物逆境胁迫响应。该研究从野生‘潘那利’番茄(Solanum pennellii Correll)中成功克隆出bHLH转录因子基因SpbHLH89(Sol Genomics登录号Sopen04g001150),采用qRT PCR分析其在干旱胁迫下的表达模式,并利用异源表达初步分析其对非生物胁迫的响应。结果表明：(1)SpbHLH89编码区包含684 bp,编码227个氨基酸,具有典型的碱性螺旋环螺旋区,主要定位于细胞核中;进化树结果显示,SpbHLH89转录因子高度保守,与拟绒毛烟草NtbHLH94(Nicotiana tomentosiformis)存在高度相似性。(2)qRT PCR结果显示,SpbHLH89在‘潘那利’番茄的茎、叶和花中均有表达,其表达量受干旱胁迫诱导。(3)SDS PAGE与Western bloting结果显示,pET 30a SpbHLH89重组蛋白大小约为31 kD。(4)在盐胁迫(400 mmol/L NaCl)和干旱胁迫(600 mmol/L甘露醇)条件下,异源表达重组蛋白的E. coli BL21(DE3)重组菌生长速度提高,说明异源表达SpbHLH89转录因子基因可提高细菌对非生物胁迫的耐受性。     

欧文氏弧菌毒力蛋白PirAB的原核表达与纯化

对虾肝胰腺坏死病的爆发造成了对虾养殖产业的严重亏损。从上海地区凡纳滨对虾中分离出1株欧文氏弧菌(Vibrio owensii SH-14),该菌株可导致凡纳滨对虾死亡,且死虾出现对虾肝胰腺坏死病的典型症状。经PCR扩增欧文氏弧菌毒力蛋白PirA与PirB对应基因序列,并将其连接到表达质粒pET-21b(pirA)和pGEX-4t-1(pirB)。通过优化诱导表达和亲和层析纯化条件,最终获得大量高纯度的目的蛋白。经冷冻干燥保存为后期抗体合成以及进一步毒力效应研究提供参考。