鹦鹉热衣原体重组主要外膜蛋白诱导小鼠免疫应答的观察

用鹦鹉热衣原体 (Chlamydiapsittaci,Cps)重组主要外膜蛋白 (RecombinantMajorOuter Membraneprotein ,r MOMP)免疫小鼠 ,观察小鼠免疫后对r MOMP和Cps菌体蛋白的免疫应答。r MOMP皮下注射免疫BALB/c小鼠 ,对照组仅注射佐剂。免疫前和第 3次免疫后 10d收集血清 ,以常规ELISA法检测抗体效价 ;MTT方法检测脾淋巴细胞对r MOMP和Cps菌体蛋白的特异性增殖反应。免疫组小鼠在免疫 38d后免疫血清中抗r MOMP的抗体效价可达 1∶2× 10 4,抗Cps菌体蛋白的抗体效价为 1∶4× 10 3 ;脾淋巴细胞对r MOMP和Cps菌体蛋白的增殖指数明显高于佐剂对照组 (p <0 .0 1,具有显著性意义。r MOMP在小鼠体内可诱导Cps特异性的体液免疫和细胞免疫应答 ,说明具有较好的免疫原性     

制备肺炎衣原体抗原片检测血清抗体

目的:探索肺炎衣原体抗原片检测血清抗体法在诊断Cpn感染中的实际应用前景。方法:应用进口肺炎衣原体(Cpn)毒株感染Hep-2细胞,分别以瑞氏-姬母萨染色、吖啶橙染色和直接免疫荧光染色等3种方法鉴定Cpn感染细胞。纯化获取大量Cpn抗原,用于制备斑点抗原片。建立微量免疫荧光染色法(MIF)检测血清抗体,诊断Cpn感染。结果:Cpn感染Hep-2细胞的最适条件是用含1μg/mL放线菌酮的维持液,在35℃、5%CO2孵箱中培养7d,并在培养的第0、3、4、5天以2600r/min离心1h,感染成功率极高。染色反应显示,瑞氏-姬母萨染色可将Cpn包涵体染成蓝紫色或红紫色;吖啶橙染色则使Cpn感染的Hep-2细胞呈现鲜明的橘红色;免疫荧光抗体染色后,在Cpn感染细胞内可见亮苹果绿色包涵体。通过斑点抗原荧光抗体染色的方法抽样检测了100份病人血清中的Cpn抗体,其中抗Cpn-IgG抗体的阳性血清共61份,阳性率为61%。与Cpn-外周血单核细胞(Cpn-PBMC)抗原片比较,阳性检出率无明显差别。结论:用Cpn感染细胞制作的Cpn斑点抗原片可用于临床检测血清Cpn-IgG抗体,且具有特异性、敏感性高的特点,但要求检测人员有一定的经验。     

Asia I口蹄疫vp2蛋白单克隆抗体的制备及单抗竞争ELISA方法的建立

制备Asia I口蹄疫病毒vp2单克隆抗体(mAb)并建立了单抗竞争ELISA方法。用纯化的Asia I型口蹄疫病毒vp2重组蛋白免疫BALB/c小鼠, 将免疫小鼠的脾细胞与骨髓瘤SP2/0细胞融合, 采用间接ELISA和有限稀释法筛选杂交瘤细胞。分别用ELISA、Western blotting检测mAb腹水的效价及其特异性。筛选到杂交瘤细胞2株, 腹水效价均在100×29以上; 以纯化后的Asia I型口蹄疫病毒vp2重组蛋白作为抗原, 利用Asia I型口蹄疫病毒vp2单抗酶标物建立了竞争ELISA方法用来检测Asia I型口蹄疫抗体。临床应用表明, 该方法与UBI公司的口蹄疫全病毒抗体检测试剂盒总符合率达89.0%, 和荷兰赛迪公司的口蹄疫病毒LPB-ELISA抗体检测试剂盒总符合率达86.5%。     

Asia Ⅰ口蹄疫vp2蛋白单克隆抗体的制备及单抗竞争ELISA方法的建立

制备Asia Ⅰ口蹄疫病毒vp2单克隆抗体(mAb)并建立了单抗竞争ELISA方法.用纯化的Asia Ⅰ型口蹄疫病毒vp2重组蛋白免疫BALB/c小鼠,将免疫小鼠的脾细胞与骨髓瘤SP2/0细胞融合,采用间接ELISA和有限稀释法筛选杂交瘤细胞.分别用ELISA、Western blotting检测mAb腹水的效价及其特异性.筛选到杂交瘤细胞2株,腹水效价均在100×2<'9>以上:以纯化后的AsiaⅠ型口蹄疫病毒vp2重组蛋白作为抗原,利用Asia Ⅰ型口蹄疫病毒vp2单抗酶标物建立了竞争ELISA方法用来检测Asia Ⅰ型口蹄疫抗体.临床应用表明,该方法与UBI公司的口蹄疫全病毒抗体检测试剂盒总符合率达89.0%,和荷兰赛迪公司的口蹄疫病毒LPB-ELISA抗体检测试剂盒总符合率达86.5%.     

肺炎衣原体ompA基因VD2-VD3区重组蛋白在血清学诊断中的初步应用

应用聚合酶联反应(PCR)技术,从肺炎衣原体Chlamydia pneumoniae的主要外膜蛋白(Major Outer Membrane Protein,MOMP)编码基因(ompA)上扩增出抗原优势表位VD2-VD3区基因,构建原核表达系统并诱导表达重组蛋白,经Ni-NTA亲和层析法纯化表达产物。间接酶联免疫吸附试验(Enzyme link immunosorbent assay,ELISA)检测人血清中特异性IgG抗体。试验表明,转化入BL21大肠杆菌的重组质粒,能表达并纯化出相对分子质量(Mr)为24KD的重组蛋白。Western blot证实重组蛋白只与Cpn MOMP mAb发生特异性反应;重组蛋白用作ELISA包被抗原检测Cpn阴阳性参比血清,特异性和灵敏度均为100%;对126位冠心病患者血清进行的检测中,该间接ELISA法与晶美公司Cpn IgG ELISA诊断试剂盒的检测结果相比,符合率达到96.3%。结果证实,制备的重组蛋白MOMPVD2-VD3具有良好的免疫活性,在Cpn血清学诊断的应用中具有较大的利用价值。     

外周血单个核细胞中肺炎衣原体的分离、培养及连续传代

开发一种分离并增殖肺炎衣原体(Cpn)的简易方法极具意义。该方法先分离血液标本中的外周血单个核细胞(PBMC),用PEG使Cpn-Ag阳性的PBMC裂解释放出Cpn,然后与人喉表皮癌细胞(Hep-2)一起离心,继续培养Hep-2细胞,再将Hep-2细胞冻融破碎后,放入到新的Hep-2细胞中进行离心,以完成Cpn的1次传代,然后以同样的方法进行2-4次传代。分别用微量免疫荧光法(MIF)及PCR法检测Hep-2细胞中的Cpn-Ag和CpnDNA,并用FITC标记的属特异性衣原体脂多糖单克隆抗体检测实验分离以及进口菌株传代后的包涵体形成单位数量。结果显示MIF法检测Cpn感染后的Hep-2细胞,其胞内Cpn-Ag强阳性;MIF法检测1次传代及2次传代的Hep-2细胞,其胞内Cpn-Ag亦均强阳性,3次传代为阳性,而4次传代为阴性;PCR法检测2次传代后CpnDNA阳性。该简化方法可以实现PBMC中Cpn的分离,分离菌株传代4次后出现退化(优于进口菌株),但该方法仍可实现进一步的培养及传代。     

HPV6L1/CtMOMP多表位融合基因在COS-7细胞的表达及其在小鼠的免疫效果观察

研究人乳头瘤病毒(human papillomavirus,HPV)6b结构蛋白L1(HPV6b L1)基因佐剂增强沙眼衣原体(Chlamydia trachomatis,Ct)主要外膜蛋白(MOMP)多表位(Ct MOMP168)DNA疫苗的免疫效果的可能性.构建pcDNA3.1(+)/HPV6b L1/Ct MOMP168融合重组质粒,转染COS-7细胞,用RT-PCR、激光共聚焦显微技术及Western印迹技术检测其表达.分别用pcDNA3.1(+)/HPV6b L1/Ct MOMP168、pcDNA3.1(+)/Ct MOMP168及pcDNA3.1(+)质粒肌肉免疫BALB/c小鼠,ELISA检测外周血中IgG及阴道分泌物中sIgA.结果表明,pcDNA3.1(+)/HPV6b L1/CtMOMP168可在COS-7细胞中表达;Ct MOMP168组和HPV6b L1/Ct MOMP168组均可刺激小鼠产生抗Ct MOMP特异性的抗体,抗体滴度随免疫次数增加而升高,且HPV6b L1/Ct MOMP168组小鼠产生的抗体滴度明显高于Ct MOMP168组(P0.05).结果提示,分子佐剂HPV6b L1与CtMOMP多表位基因融合能够显著增强Ct MOMP多表位DNA疫苗的体液免疫应答.     

抗卵清蛋白单克隆抗体杂交瘤细胞株的制备

为制备分泌抗卵清蛋白的杂交瘤细胞,以高纯度的卵清蛋白抗原免疫BALB/c小鼠,取其脾脏细胞和Sp2/0骨髓瘤细胞融合,获得杂交瘤细胞,用ELISA间接法检测上清液中的抗卵清蛋白抗体效价,经3次单克隆化筛选,获得5株分泌抗卵清蛋白抗体的杂交瘤细胞株。     

抗人B7-H1单克隆抗体的制备和鉴定

目的:采用杂交瘤技术制备抗人B7-H1单克隆抗体,并对其进行鉴定。方法:经抗原免疫的小鼠脾细胞与小鼠骨髓瘤细胞以常规方法融合;用间接ELISA法筛选分泌抗体的杂交瘤细胞株;阳性克隆用有限稀释法获得稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株;扩增杂交瘤细胞注射进小鼠腹腔后制备腹水;纯化腹水中的单克隆抗体并对其亚型进行鉴定;用间接ELISA法测抗体效价;将肺癌组织制成石蜡切片,用抗人B7-H1抗体进行免疫组化染色。结果:获得1株稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株,所分泌的单抗类型为IgG1;抗体效价为1×108,纯化后的抗体含量为6.76g/L;免疫组化实验中,单抗可与肺癌组织表面的B7-H1蛋白特异地结合。结论:制备了人B7-H1单克隆抗体,为B7-H1检测试剂盒的研制奠定了基础。     

口蹄疫病毒单克隆抗体的制备及检测应用

用纯化的口蹄疫病毒(Footandmouthdiseasevirus,FMDV)免疫BALB/C小鼠,将免疫鼠的脾细胞与SP2/0骨髓瘤细胞融合,采用有限稀释法进行克隆,经筛选获得多株能稳定分泌抗FMDV单抗的杂交瘤细胞株。选择其中一株(2G12)用于下列实验,其细胞培养上清液的效价是1:256,腹水效价是1:1280;以自行制备的兔抗FMDV高免血清IgG为捕获抗体包被酶联免疫吸附试验微量反应板,以单抗2G12为第二抗体,建立了快速检测FMDV抗原的双抗体夹心ELISA,该方法能检出90ng病毒,而且只与FMDV发生特异性反应,与猪瘟病毒(HCV)、猪蓝耳病病毒(PRRSV)、伪狂犬病毒(PRV)、猪细小病毒(PPV)和乙脑病毒(JEV)均不发生反应。本研究为检测口蹄疫病毒抗原提供了灵敏和特异的方法。     

Cpn20: Siamese twins of the chaperonin world

The chloroplast cpn20 protein is a functional homolog of the cpn10 co-chaperonin, but its gene consists of two cpn10-like units joined head-to-tail by a short chain of amino acids. This double protein is unique to plastids and was shown to exist in plants as well plastid-containing parasites. In vitro assays showed that this cpn20 co-chaperonin is a functional homolog of cpn10. In terms of structure, existing data indicate that the oligomer is tetrameric, yet it interacts with a heptameric cpn60 partner. Thus, the functional oligomeric structure remains a mystery. In this review, we summarize what is known about this distinctive chaperonin and use a bioinformatics approach to examine the expression of cpn20 in Arabidopsis thaliana relative to other chaperonin genes in this species. In addition, we examine the primary structure of the two homologous domains for similarities and differences, in comparison with cpn10 from other species. Lastly, we hypothesize as to the oligomeric structure and raison d’être of this unusual co-chaperonin homolog. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

衣原体分泌性蛋白研究进展

衣原体分泌性蛋白是由衣原体基因编码并分泌到宿主细胞胞浆中的具有酶活性蛋白,研究最多的是蛋白酶体样活性因子即CPAF。CPAF能抑制IFN-γ诱导的MHC分子的表达、裂解角蛋白-8并通过裂解宿主细胞唯BH3域蛋白参与抗凋亡作用。2005年又发现两种肺炎嗜衣原体的分泌性蛋白CPn0796和CPn0797,但对其研究不多。     

Twintrons are not unique to the Euglena chloroplast genome: structure and evolution of a plastome cpn60 gene from a cryptomonad

Introns within introns (twintrons) are known only from the Euglena chloroplast genome. Twintrons are group II or III introns, into which another group II or III intron has been transposed. In this paper we describe a non-Euglena twintron structure within a plastid-encoded chaperone gene (cpn60) of the cryptomonad alga Pyrenomonas salina. In addition, the evolutionary relationships between members of the Cpn60 protein family are determined. Our findings permit the inclusion of cryptomonad plastomes in phylogenetic studies of intron evolution and present further evidence for the origin of modern plastids from a cyanobacterial ancestor.This paper is dedicated to Prof. Dr. Peter Sitte on the occasion of his 65th birthday     

肺炎衣原体蛋白激酶Cpn0148的克隆表达和定位

目前认为肺炎衣原体(Chlamydia pneumonia, Cpn)除导致呼吸道疾病外, 也是与冠心病相关的重要病原体。作为一种细胞内寄生的病原菌, Cpn激活宿主细胞信号通路, 维护其在细胞内生长代谢, 并导致疾病。肺炎衣原体基因Cpn0148可编码真核细胞样的丝/苏氨酸蛋白激酶, 利用PCR技术扩增全长Cpn0148 ORF, 将其定向插入pGEX-6p原核表达载体, 在大肠杆菌XL-1blue中表达, 测序显示Cpn0148 ORF全长1860 bp, 编码619个氨基酸, 分子量大约70 kD,     

OsCpn60α1, encoding the plastid chaperonin 60α subunit, is essential for folding of rbcL

Chaperonins are involved in protein-folding. The rice genome encodes six plastid chaperonin subunits (Cpn60) — three α and three β. Our study showed that they were differentially expressed during normal plant development. Moreover, five were induced by heat stress (42°C) but not by cold (10°C). The oscpn60α1 mutant had a pale-green phenotype at the seedling stage and development ceased after the fourth leaf appeared. Transiently expressed OsCpn60α1:GFP fusion protein was localized to the chloroplast stroma. Immuno-blot analysis indicated that the level of Rubisco large subunit (rbcL) was severely reduced in the mutant while levels were unchanged for some imported proteins, e.g., stromal heat shock protein 70 (Hsp70) and chlorophyll a/b binding protein 1 (Lhcb1). This demonstrated that OsCpn60α1 is required for the folding of rbcL and that failure of that process is seedling-lethal.     

An exceptionally stable Group II chaperonin from the hyperthermophile Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus (Pf) grows optimally at 100 °C and encodes single genes for the Group II chaperonin (Cpn), Pf Cpn and α-crystallin homolog, the small Heat shock protein (sHsp). Recombinant Pf Cpn is exceptionally thermostable and remained active in high ionic strength, and up to 3 M guanidine hydrochloride (Gdn-HCl). Pf Cpn bound specifically to denatured lysozyme and ATP addition resulted in protection of lysozyme from aggregation and inactivation at 100 °C. While complexed to heat inactivated lysozyme, Pf Cpn showed enhanced thermostability and ATPase activity, and increased the optimal temperature for ATPase activity from 90 to 100 °C. Protein substrate binding also stabilized the 16-mer oligomer of Pf Cpn in 3 M Gdn-HCl and activated ATPase hydrolysis in 3-5 M Gdn-HCl. In addition, Pf Cpn recognized and refolded the non-native lysozyme released from Pf sHsp, consistent with the inferred functions of these chaperones as the primary protein folding pathway during cellular heat shock.     

Alteration of Cpn60 expression in pancreatic tissue of rats with acute pancreatitis

The expression of heat-shock protein 60 (also known as chaperonin 60, Cpn60) in experimental acute pancreatitis (AP) is considered to play an active role in the prevention of abnormal enzyme accumulation and activation in pancreatic acinar cells. However, there are controversial results in the literature regarding the relationship between the abnormality of Cpn60 expression and AP onset and development. The purpose of this study was to investigate the alternations of Cpn60 expression and the relationship between the abnormal expression of Cpn60 and AP progression in rat severe acute pancreatitis (SAP) models. In this report, we induced SAP in Sprague–Dawley (SD) rats by reverse injection of sodium deoxycholate into the pancreatic duct, and examined the dynamic changes of Cpn60 expression in pancreatic tissues from different time points and at different levels with techniques of real-time PCR, western blotting, and immunohistochemistry. At 1 h after SAP induction, the expression of Cpn60 mRNA in the AP pancreatic tissues was higher than those in the sham-operation group and normal control group, but decreased sharply as the time period was extended, and there was a significant difference between 1 h and 10 h after SAP induction (p < 0.05). In the AP process, Cpn60 protein expression showed transient elevation as well, and the increased protein expression occurred predominantly in affected, but not totally destroyed, pancreatic acinar cells. As AP progressed, the pancreatic tissues were seriously damaged, leading to a decreased overall Cpn60 protein expression. Our results show a complex pattern of Cpn60 expression in pancreatic tissues of SAP rats, and the causality between the damage of pancreatic tissues and the decrease of Cpn60 level needs to be investigated further. Xue-Li Li and Kun Li contributed equally to this work.     

Three GroEL homologues from Rhizobium leguminosarum have distinct in vitro properties

The GroEL molecular chaperone of Escherichia coli and its cofactor GroES are highly conserved, and are required for the folding of many proteins. Most but not all bacteria express single GroEL and GroES proteins. Rhizobium leguminosarum strain A34 encodes three complete operons encoding homologues to GroEL and GroES. We have used circular dichroism and measurement of ATPase activity to compare the stabilities of these chaperonins after expression in and purification from E. coli. Significant differences in the stabilities of the proteins with respect to denaturant and temperature were found. The proteins also differed in their ability to refold denatured lactate dehydrogenase. This study, the first to compare the properties of three different GroEL homologues from the same organism, shows that despite the high degree of similarity between different homologues, they can display distinct properties in vitro.