Monoclonal antibodies against HPV11 virus-like particles: functional characteristics and application on quality assessment

目的分析和鉴定抗HPV11病毒样颗粒(virus-like particle,VLP)鼠源单克隆抗体的性质,筛选性质和生物学活性较优的抗体,并初步应用于抗原或疫苗的质量分析。方法分别利用间接ELISA法和Western blot对HPV11的22株单克隆抗体的亚类、与HPV11 VLP的结合能力和构象敏感性进行检测;采用血凝抑制实验对单克隆抗体的血凝抑制活性进行分析;运用基于假病毒的抗体中和实验对单克隆抗体的中和活性进行鉴定,选出中和活性高的单抗进行两两配对,采用双抗夹心ELISA法捕获单抗并筛选合适的配对双抗。结果对22株单抗的性质进行了详细和完整的鉴定,并根据构象敏感性进行排序,筛选出6株型别特异、结合活性强且中和活性高的单抗(2A2、4A1-3、16G7、14A6、9C12和19C7);成功建立了基于单抗的双抗夹心(14A6∶Ag∶9C12-HRP)ELISA定量分析方法。结论获得了较全面的HPV11 VLP单抗性质信息,建立了重组HPV11抗原质量分析的双抗夹心ELISA法,为HPV11抗原的生命周期管理或尖锐湿疣疫苗的研发、工艺优化、产品放行和稳定性研究等提供了技术支持。Objective To quantitatively analyze the characteristics of a panel of murine anti-human papillomavirus( HPV) 11 L1-derived virus-like particle( VLP) monoclonal antibodies( m Abs) and establish the m Ab-based methods for antigen quality analysis. Methods A panel of 22 murine anti-HPV11 m Abs were characterized in details with their isotype,and binding affinity,conformational sensitivity were examined quantitatively in the direct binding ELISA and Western blot. The hemagglutination inhibition activity of m Abs were identified using the hemagglutination inhibition assay and the pseudovirus( Ps V) neutralization efficiency were examined quantitatively using the Ps V-based neutralization assay. The type-specific, highly conformational sensitive and neutralizing m Abs were selected to be used in the sandwich ELISA assay. Results Based on the quantitative and semi-quantitative results,six type-specific,highly conformational sensitive and neutralizing m Abs( 2A2,4A1-3,16G7,14A6,9C1 and 19C7) were identified. These m Abs,along with 10D6 were screened as the capture m Ab or as the detection m Ab in the sandwich ELISA. Conclusion The binding affinity,conformational sensitivity and neutralization efficiency of anti-HPV11 m Abs were characterized in details. A m Ab-based sandwich ELISA assay( 14A6∶Ag∶9C12-HRP) were developed,which could be used in the in vitro potency analysis of HPV11 VLP-based vaccine.重大新药创制(2015ZX09101034

量子环面上一类导子李代数的结构和自同构群

本文研究量子环面上的一类导子李代数, 它包含了∗ +, −. /, /0 1+ 23 代数及其4 类似( 首先证明了这类导子李代数之间的同构一定是分次同构, 并进一步给出了代数同构的充要条件及同构映射的具体表达式, 最后确定了该类李代数的自同构群 (国家自然科学基金（NO.10371100) 福建省教育厅基金（NO.04303） 漳州师范学院科研基金(NO.03003

胶原特性及其制备方法研究进展

本文阐述了胶原、明胶和胶原多肽三者的概念和不同点,介绍了胶原的结构以及Ⅰ型胶原和Ⅱ型胶原的差异,总结了胶原的七种制备方法,并对各种制备方法的优缺点进行分析,为水产动物资源制备胶原提供参考。福建省海洋生物增养殖与高值化利用重点实验室(361013);;福建省海洋生物资源开发利用协同创新中心(361013

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目的 研究高危型人乳头瘤病毒（HR-HPV）感染相关的宫颈病变程度与抗人乳头瘤状病毒（HPV）抗体水平的相关性,探讨HPV致病的免疫学问题。方法 选取2012年1月-2013年6月在该院妇产科门诊就诊的女性中选取60例病理确诊为女性宫颈病变（CINⅠ及以上）,且宫颈脱落细胞的DNA检测为HR-HPV阳性者为研究组,根据组织学检测结果将研究组进一步分为低度病变组30例（CINⅠ）和高度病变组30例（CINⅡ或Ⅲ）。对照组为经核酸检测无HPV感染且病理诊断未见癌前病变的门诊患者60例,按年龄进一步分为低年龄组和高年龄组。低年龄组和高年龄组与低度病变组和高度病变组的年龄差异无统计学意义（P〉0.05）。分别抽取研究组及对照组外周血,检测其抗HR-HPV L1 Ig G抗体及中和抗体滴度,比较研究组与对照组抗体阳性率及抗体滴度水平的差别。结果 研究组血清中的抗HR-HPV L1 Ig G抗体阳性率及抗体滴度均高于对照组（P〈0.05）;低度病变组患者血清中的抗HR-HPV的中和抗体滴度高于相应对照组（P〈0.05）,而高度病变组的抗HR-HPV的中和抗体阳性率及抗体水平与相应对照组比较差异无统计学意义（P〉0.05）。结论 宫颈病变患者血清中抗HR-HPV L1 Ig G抗体升高,说明HPV感染可引起机体的体液免疫反应。低度病变组的中和抗体水平相对较高,可能是机体产生的中和抗体阻止了病毒的进一步感染。高度病变组的Ig G抗体和中和抗体均低下,说明进入CINⅡ/Ⅲ后HPV已经逃避了免疫系统的监视。基金项目：厦门市科技局项目（3502220124050）;2011年福建省科技计划项目资助计划,青年创新项目（20111）00310517

抗H5亚型禽流感病毒单链抗体在毕赤酵母中的分泌型表达和生物活性分析

在本实验室研制出的多株针对H5N1亚型禽流感血凝素单抗中,13D4单抗对所有H5亚型病毒均有血凝抑制和中和活性,具有特异性高、反应性强和识别广的特点,且在小鼠实验中显示了对各种代表株禽流感的感染和发病均具有良好的治疗效果。在此研究基础上,本实验通过基因工程构建含有13D4单链抗体(scFv)基因的毕赤酵母表达载体,实现目的蛋白的分泌性表达和纯化。经过竞争法和血凝抑制检测其活性,表明获得的单链抗体具有与原始鼠源抗体相近的反应活性和相同的识别表位。H5N1广谱中和单抗13D4的单链抗体的成功构建,为进一步研制针对H5N1禽流感病毒的治疗性抗体奠定了基础

Characterization and immunoprotective effect of SjIrV1,a 66 kDa calcium-binding protein from Schistosoma japonicum

钙结合蛋白是日本血吸虫生长发育不可缺少的蛋白,具有非常广泛而重要的功能。在课题组日本血吸虫体被表膜蛋白研究基础上,利用PCr技术克隆了中国大陆株日本血吸虫66 kdA钙结合蛋白(SJIrV1)编码基因的CdnA序列,blAST分析与菲律宾株日本血吸虫SJIrV1 CdnA编码序列一致,荧光定量PCr分析表明该基因在童虫和成虫期不同发育阶段均有表达,其中在35 d和42 d成虫中表达量较高,在42 d雌虫中该基因表达水平远高于42 d雄虫。构建重组表达质粒PET28A(+)-SJIrV1,在大肠杆菌中成功诱导表达,重组蛋白主要以可溶性形式存在,通过高效液相色谱法(rP-HPlC)以及串联质谱法(MS/MS)鉴定所获蛋白为目的蛋白SJIrV1。蛋白质印迹(WESTErn blOTTIng)分析结果显示重组蛋白能被感染日本血吸虫鼠血清和免疫鼠血清所识别,SJIrV1蛋白在虫体各发育阶段中均表达。免疫荧光染色实验观察表明SJIrV1主要分布在日本血吸虫成虫的表膜。应用重组蛋白免疫bAlb/C小鼠后,免疫鼠血清中检测到较高水平的特异性Igg、Igg1和Igg2A抗体。结果表明SJIrV1可能在日本血吸虫的生长发育过程中起着重要作用。Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum.Based on our primary study on tegument surface proteins of S.japonicun,a cDNA encoding a 66 kDa calcium-binding protein of S.japonicum(Chinese strain) was cloned,sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S.japonicum.The expression of SjIrV1 were detected by Real-time PCR,using cDNA templates isolated from 7,14,21,28,35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages,and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms.The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a(+) vector and transformed into competent Escherichia coli BL21 for expression.The recombinant protein was purified using a Ni-NTA purification system,and confirmed by high performance liquid chromatography(RP-HPLC) and tandem mass spectrometry(MS/MS).Western blotting analysis showed that recombinant SjIrV1(rSjIrV1) could be recognized by the S.japonicum infected mouse serum and the mouse serum specific to rSjIrV1,respectively.Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms.ELISA test revealed that IgG,IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice.The study suggested that rSjIrV1 might play an important role in the development of S.japonicum.国家自然科学基金(No.31172315); 上海科技发展基金(No.12140902700); 中国博士后科学基金(No.2012M510630)资助~

Identification and preliminary analysis of a novel full-length cDNA encoding retinoid X receptor 2 from Schistosoma japonicum

通信作者 E-mail: [email protected][中文文摘]目的克隆编码日本血吸虫视黄酸X受体2(SjRXR2)蛋白的全长cDNA,并对其进行初步研究。方法利用cDNA末端快速扩增技术(RACE)获得SjRXR2蛋白全长编码cDNA。利用生物信息学技术,对基因结构进行初步分析。利用实时荧光定量(Real time)PCR技术对该基因在日本血吸虫不同时期虫体中的转录情况进行分析。应用在线抗体表位预测软件获得SjRXR2配体结合区抗原性较强的一个多肽序列,合成该多肽片段,并免疫小鼠制备抗血清。利用Western blot技术分析该蛋白在日本血吸虫中的表达。结果采用RACE技术成功获得了SjRXR2蛋白全长编码cDNA,总长度为5 960bp,其完整开放阅读框为4 308 bp,编码1 435个氨基酸,预测分子量为159 kDa。生物信息学分析表明该基因编码的蛋白质序列具有核受体家族2的典型结构域特征,且与曼氏血吸虫RXR2有较高的相似性。Real time PCR分析表明,该基因在21、42 d龄日本血吸虫虫体内有较高的转录水平。Western blot分析表明,小鼠SjRXR2多肽免疫血清可特异性识别日本血吸虫虫体150 kDa蛋白。结论成功获得了编码SjRXR2蛋白的全长 cDNA，并制备了针对该蛋白的特异性多克隆抗体，为进一步研究该蛋白的功能奠定了基础。 .国家自然科学基金（31172315）；公益性行业（农业）科研专项（20090303036