从噬菌体随机肽库中筛选SARS病毒N蛋白表位模拟肽的研究

目的获得具有生物学活性的SARSN蛋白的模拟肽。方法以抗SARS病毒N蛋白的单克隆抗体作为固相筛选分子,免疫筛选噬菌体随机十二肽库,并采用夹心ELISA、竞争抑制试验鉴定阳性克隆。结果经噬菌体富集后,从随机筛选的各46个克隆中得到N蛋白的2个不同表位,即由ELISA鉴定出单克隆抗体C00835个阳性克隆,确定氨基酸序列GXLXTPXXXXGT为SARSN蛋白的一个表位;单克隆抗体C03941个阳性克隆,确定氨基酸序列TTLPXXXXAXXX为SARS病毒N蛋白的另一个表位。结论用噬菌体随机12肽库成功筛选得到SARSN蛋白的2个不同模拟表位,为用SARS表位去探索其病毒的结构及疫苗的研制创造了条件。     

Encapsidating artificial human papillomavirus-16 mE7 protein

Background Human papillomaviruses （HPVs） can infect squamous or mucosal epithelia and cause cervical cancer or genital warts. Coinfection with multiple HPV types is a common finding of many epidemiological studies. Therefore, it is necessary to develop a vaccine, which can eradicate established HPV infections and prevent other HPV infections. In this study, we generated chimeric virus like particles （cVLPs） composed of HPV-6b L1, HPV-6b L2 and one artificial HPV-16 mE7 proteins. Methods The artificial HPV-16 mE7 gene was designed by codon modification, point mutation and gene shuffling then chemically synthesized and subcloned behind HPV-6b L2. HPV-6b L1 and L2-mE7 were expressed in insect cells by using Bac-to-Bac system. The generated cVLPs were purified by CsCI gradient ultracentrifuge and analyzed by immunoblot, electron microscope and haemagglutination assay. Results The HPV-6b L1 and L2-mE7 proteins were well expressed in insect cells and could selfassemble into cVLPs, whose diameter was about 55 nm and similar to that of HPV-6b L1/L2 VLPs. Intact cVLPs could be recognized by H6.M48 neutralizing monoclonal antibody and HPV-6b L2 polyclonal antibody, while the denatured cVLPs, but not the intact cVLPs, were reactive to HPV-16 E7 polyclonal antibody. HPV-6b LI/L2-mE7 cVLPs haemaggiutinated mouse erythrocytes as efficiently as HPV-6b L1/L2 VLPs did. Conclusions The insertion of the 158 amino acid HPV-16 mE7 protein behind L2 did not disrupt the correct assembling of cVLPs. The morphological characteristics and haemagglutinating activity of cVLPs were similar to those of HPV-6b LI/L2 VLPs. The cVLPs retained conformational B cell epitopes of HPV-6 VLPs and HPV-16 mE7 protein had an internal location in the cVLPs. Therefore, large modified E7 protein with higher immunogenicity could be incorporated into cVLPs by fusing to the C-terminus of L2, which would help to improve the therapeutic effects of LI/L2-E7 cVLPs.     

共表达HPV16L1、L2、E67蛋白的重组非复制型痘苗病毒的构建

目的：构建共表达人乳头瘤病毒16型(HPV16)L1、L2、E67蛋白的非复制型重组痘苗病毒人用疫苗株。方法：以痘苗病毒为载体、利用同源重组技术筛选共表达HPV16L1、L2、E67蛋白的重组痘苗病毒并对其进行鉴定。结果：该病毒在CEF细胞上连续传至第15代,经斑点杂交结果表明重组病毒基因组中有L1、L2、E6、E7基因插人;经Western blot检测,重组病毒能稳定表达HPV16L1、L2、E67蛋白。结论：非复制型重组痘苗病毒NTVJE67CKL IL2可作为预防和治疗HPV16相关肿瘤及其癌前病变的候选疫苗。     

Immune response to plasmid DNA encoding HPV16-L1 protein

Objective To test the immunogenicity of recombinant plasmid DNA containing human papillomavirus type 16-L1 (HPV16-L1) coding sequence of mice.Methods The HPV16-L1 encoding sequence was generated by polymerase chain reaction (PCR), and inserted into TA cloning vector PCR Ⅱ, then cloned in the eukaryotic expression vector pcDNA3.1 with CMV promoter. The recombinant plasmid DNA pcDNA-L1 was transferred into Cos-7 cells and used to immunize BALB/c mice via muscular injection. The expression of HPV16-L1 in transferred cells was identified by immunospot and immunocytochemistry, which tested specific anti-HPV16-L1 antibody in the serum of immunized mice. Results Using the immunospot technique, we found L1 protein expression in pcDNA-L1 transferred cells. The immunocytochemistry studies demonstrated that the L1 protein was located in nuclei. In immunized mice, specific anti-HPV16-L1 antibodies could be detected by immunospot and immunocytochemistry 28 days after the first immunization and last at least 41 days. Conclusions We constructed HPV16-L1 eukaryotic expressing plasmid whose DNA could induce immuno-humoral response in mice. This observation will be helpful in designing HPV16 prophylactic vaccine.     

HPV16L1重组蛋白在甲醇营养型酵母菌中的诱导表达及鉴定

目的 ＨＰＶ１６Ｌ１重组蛋白的表达为研制ＨＰＶ１６Ｌ１预防性蛋白疫苗及诊断试剂盒打下基础。方法 ｐＰＬＣ３．５－ＨＰＶ;１６Ｌ１／ＧＳ１１５阳笥菌株,经０．５％甲醇诱导,运用ＳＤＳ－ＰＡＧＥ电泳检测Ｌ１蛋白;用鼠抗ＨＰＶ１６Ｌ１单克隆抗体,经Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ检测蛋白的特异笥;在透射电下观察类病毒颗粒（ＶＬＰｓ）结果 ＳＤＳ－ＰＡＧＥ检测结果表明,在５５ＫＤ处有诱导蛋白带的出现;经Ｗ     

HPV L1病毒样颗粒疫苗诱导机体免疫反应的研究现状

HPV感染与宫颈癌发展密切相关,HPV病毒样颗粒(VLP)在体外由病毒的主要衣壳蛋白L1自发装配而成。HPV VLP成为候选疫苗,它能很有效的减少感染并且诱导很强的抗病毒免疫。免疫后产生的中和抗体具有型特异性,接种VLP后也能产生细胞免疫,疫苗可诱导出至少持续5年的高滴度抗L1VLP抗体,比自然感染高10倍。疫苗诱导的长期保护效应通常是免疫记忆的特征。     

人乳头瘤病毒基因工程疫苗的基础实验研究

人乳头瘤病毒(HPV)16型感染与宫颈癌的发生、发展密切相关,故研制HPW16型基因工程疫苗对预防HPV感染、控制宫颈癌的发生有重要意义。本研究构建HPV16 L1基因工程菌并进行了基础实验研究。首先采用PCR技术从3例宫颈癌组织中扩增和克隆了HPV16L1基因片段并进行测序。进而构建了pPIC3.5～HPV16L1真核表达工程菌、pQE31～HPV16L1和pGEX4T-HPV16LI原核表达工程菌及杆状病毒-昆虫细胞表达系统。经SDS-PAGE、Westem blot和电镜等手段证明了工程菌表达HPV16 L1蛋白的分子特性和免疫原性,而且,重组菌表达的HPV16 L1可以形成类病毒颗粒(VLP)。经免疫BALB/C小鼠可获得高效价特异血清,证实重组HPV16 L1蛋白具有良好的免疫原性。为进一步研制临床应用疫苗及血清学诊断试剂提供了实验基础。     

单克隆抗体识别的癌胚抗原决定簇及其生化特性

本文应用系列抗CEA及相关抗原的单抗系列,探测了其抗原决定簇、生化特性及抗体反应的特异性。并以物理、化学因素处理、鉴定了抗原的化学稳定性及相关结构。以亚细胞膜结合-微量固相放免分析试验,分析了此系列单抗与结直肠癌组织细胞反应的限定性。     

丙型肝炎病毒包膜蛋白E2抗原模拟表位的筛选和鉴定

目的：筛选丙肝病毒（HCV）包膜蛋白（E2）特异性噬菌体模拟表位。方法：以抗－HCV E2的单克隆抗体作为固相筛选分子，对噬菌体同七肽库进行5轮“吸附－洗脱－扩增“的筛选过程，随机挑取50个克隆，经酶联免疫吸附法（ELISA）鉴定、交叉反应以及竞争抑制性实验，最后对所选克隆进行DNA序列分析，以确定HCV E2抗原的模拟表位。结果：经噬菌体富集后，得到12个阳性克隆，确定氨基酸序列XXPXYXW为HCV E2的模拟表位。结论：用噬菌体七肽库成功筛选得到HCV E2的模拟表位，为开展用HCV模拟表位探索HCV的防治研究创造了条件。     

HPV vaccine: Current status and future directions

HPV Vaccine was introduced to prevent cervical cancer known to be caused by infection with one or more of the high risk subtypes of the Human papilloma virus (HPV). Since introduction, trials have proven its efficacy in preventing Cervical intraepithelial neoplasia (CIN) beyond doubt and its effectiveness in preventing cervical cancer though presumptive is reasonably certain as per mathematical modelling. It also prevents other HPV related anogenital and oropharyngeal malignancies in both sexes. HPV vaccines have courted many controversies related to its efficacy, safety, ideal age of vaccination, use in HPV infected individuals and use in males. The currently available vaccines are based on L1 Viral like particles (VLP) and hence highly species specific, thermolabile, costly and are purely prophylactic. The quest for a cheaper, thermostable and broad spectrum vaccine has led to many newer prophylactic vaccines. Therapeutic vaccines were born out of the inescapable necessity considering high HPV related morbidity projected in the non HPV naïve population. Therapeutic vaccines would immediately reduce this burden and also help in the management of HPV related cancers alone or as part of combination strategies. Ongoing research is aimed at a total control over HPV related malignancies in the near future.     

Human papillomavirus DNA and virus-encoded antigens in cervical carcinoma

The present study was undertaken to evaluate the prevalence of HPV in formalin-fixed, paraffin-embedded cervical carcinoma tissues using PCR followed by non-radioactive Southern hybridization with type-specific oligonucleotides for HPV 16 and 18. In addition, the tissue sections were immunohistochemically screened with two monoclonal antibodies, for expression of HPV 16 L1 and HPV 18 E6 proteins. A total of 57 of 60 cervical carcinomas (95.0%) were found with HPV using both techniques. HPV 16 and HPV 18 were present in equal proportions. Results of both DNA hybridization and immunohistochemistry were in agreement for the majority of the cases. HPV 16 and 18 DNA and virus-encoded antigens, L1 and E6 were found highly prevalent in these cervical carcinomas. Due to the high prevalence of HPV with cervical carcinoma in Malaysia, the implementation of routine diagnosis for the virus in cervical biopsies would be clinically useful.     

抗抗总黄曲霉毒素单抗F(ab’)2多克隆抗体的制备及特性

目的:以抗总黄曲霉毒素单抗2G2株F(ab’)2片段作为免疫原制备抗抗总黄曲霉毒素单抗独特型多克隆抗体。方法:制备抗总黄曲霉毒素单抗2G2株F(ab’)2片段,分别用F(ab’)2片段和F(ab’)2-KLH免疫日本大耳白兔,制备抗抗总黄曲霉毒素单抗独特型多克隆抗体。采用ELISA检测该抗体替代黄曲霉毒素B1(AFB1)的情况,并分析与其他单克隆抗体的交叉反应情况。用该多克隆抗体免疫BALB/C小鼠,采用间接非竞争ELISA检测小鼠血清,鉴定该抗体的亚型。结果:由2G2株F(ab’)2片段所得的抗抗总黄曲霉毒素单抗独特型多克隆抗体纯化后,替代游离AFB1达到20%、50%和80%竞争抑制时的质量浓度分别为1、4和25mg/L,分别相当于0.47、2.74和16.00μg/L游离AFB1;该抗体替代AFB1包被抗原达到20%、50%和80%竞争抑制时的质量浓度分别为1、4和19mg/L,分别相当于0.33、1.35和5.45μg/L游离AFB1。小鼠生物鉴定显示该多克隆抗体为Ab2β型。结论:成功制备了抗抗总黄曲霉毒素单抗独特型多克隆抗体,为研制AFB1无毒检测试剂盒提供了依据。     

从随机多肽库中筛选日本血吸虫抗原模拟表位诱导保护性免疫的研究

目的 从噬菌体随机肽库中筛选出能模拟日本血吸虫抗原表位的短肽分子，并测定其作为疫苗候选分子的潜能。方法 分别用正常及感染日本血吸虫的东方田鼠血清筛选在丝状噬菌体表面表达的随机十二肽库。经三轮亲和筛选后，随机挑取噬菌体克隆用ELISA检测其与日本血吸虫抗血清的反应。用混合噬菌体免疫小鼠并进行攻击感染。结果 随机挑取的12个克隆中，感染东方田鼠血清来源的噬菌体有10个克隆为阳性，正常血清来源的噬菌体有7个克隆为阳性。用感染东方田鼠血清筛到的噬菌体免疫小鼠诱导了部分保护作用（22．6％）及较高的肝卵减少率（68．9％）。然而，正常东方田鼠血清筛到的噬菌体没有保护效果。结论 感染及正常东方田鼠血清筛到的模拟表位具有免疫原性，提示随机肽库技术在日本血吸虫疫苗研制中有其潜在价值。     

特异性SARS-CoV N蛋白单克隆抗体的制备及抗原表位初步分析

目的：用分段表达纯化的SARS冠状病毒（SARS-CoV）的核衣壳蛋白（nucleocapsid N蛋白）制备针对该蛋白不同区域抗原表位的高特异性单克隆抗体（McAb）,初步定位单克隆抗体识别表位所在区域。方法：用分段表达纯化的SARS～CoV的N蛋白（分别记为N1蛋白、N2蛋白）分别免疫Balb／c小鼠制备McAb,通过检测其亚类、效价及相对亲和力鉴定McAb的生物学特性,以Western blot鉴定单克隆抗体特异性,并对McAb结合表位进行初步分析。结果：筛选出7株抗SARS-CoV N1蛋白及2株抗SARS-CovN2蛋白的单克隆抗体杂交瘤细胞株,IgG亚类鉴定6株为IgG1,2株为IgG2b,1株为IgG3,腹水效价10^5以上;亲和常数达10^8以上。Western blot证实所获的单克隆抗体可与SARS-CoVN蛋白发生特异性反应。ELISA相加实验结果显示2株N1 McAb识别相同的抗原表位,其余均识别不同的抗原表位。结论：通过分段表达纯化的SARS-CoVN蛋白而制备的特异性针对SARS-CoV N蛋白不同区域抗原表位的单克隆抗体中,N1单抗识别N蛋白N端（1-549bp）,N2单抗识别N蛋白C端（496～1269bp）,可初步定位单克隆抗体识别表位所在区域。     

贺州市妇女人乳头瘤病毒感染疫情分析

目的分析贺州市妇女人乳头瘤病毒（HPV）感染基因型与年龄分布特点,为HPV预防性疫苗研发及引进提供数据备案。方法采用PCR体外扩增和DNA反向点杂交相结合的DNA芯片技术（PCR-RDB）,对2 092份宫颈分泌物样本的HPV基因亚型检测结果进行回顾性分析,了解本地的HPV感染疫情;同时将受检样本按年龄分为16～25岁、26～35岁、36～45岁、46～55岁、〉55岁五组,分析比较不同组之间的感染特点。结果 2092例样本中共检出阳性590例,阳性率为28.2%,感染的主要基因型前三位是HPV16、58、52。各年龄组感染阳性率依次为66.9%、23.0%、23.0%、30.0%、28.9%;大多数患者只感染单一基因亚型（75%）,感染双重以上基因型的比率在各年龄组的依次是43.4%、19.9%、18.9%、22.1%、21.6%。结论贺州市的HPV流行亚型主要为16、58、52型,各年龄段HPV亚型分布有一定差别,青少年为我市的HPV感染高危人群,青少年人群有接受HPV预防疫苗的充分必要。     

CONSTRUCTION AND IMMUNOGENICITY OF HUMAN PAPILLOMAVIRUS TYPE 6B L1 RECOMBINANT PLASMID

Objective To construct a DNA vaccine as a prophylactic model to prevent condyloma acuminatum and detect its immunogenicity in mice. Methods The major capsid protein (L1) gene of human papillomavirus (HPV) 6b was inserted into an eukaryotic expression plasmid (pcDNA3.1). The recombinant plasmid was transfected into COS-7 cells. Western blot were performed to detect whether L1 protein can be expressed in eukaryotic cells. Eighteen female BALB/c mice were tested for immunogenicity study. Results The recombinant plasmid (pcDNA3. 1-HPV6bL1) was verified as HPV6b L1 gene by sequencing. Western blot showed specific strip. Anti-L1 protein antibodies could be detected in the mice‘s sera inoculated with pcDNA3.1-HPV6bL1. Similarly, IL-4, IL-2, and IFN-γ were increased in the same mice. Conclusion HPV6b L1 recombinant plasmid was constructed successfully which had immunogenicity for BALB/c mice. It provided experimental evidence for the research of DNA vaccine of condyloma acuminata.     

HPV16L1-E7重组腺病毒rAd5HPV16L1-E7的构建及克隆表达

目的研制人乳头瘤病毒16型(HPV16)L1-E7重组腺病毒,以期获得防治宫颈癌的重组腺病毒减毒活疫苗和嵌合病毒样颗粒疫苗。方法以HPV16型野毒株HPV16-114／K为模板,利用PCR克隆技术构建可表达HPV16主要衣壳蛋白L1(1-301aa)和转化蛋白E7(1-60aa)融合基因的重组质粒pUC19L1-E7,HPV16L1-E7DNA片段经质粒pBSLl-E7转入腺病毒Ad5穿梭质粒pCA14L1-E7,与腺病毒质粒pBHGl0共转染293细胞,制备重组腺病毒rAd5HPV16L1-E7,密度梯度超迷离心纯化HPV16嵌合L1-E7VLP。结果成功构建了HPV16L1-E7重组质粒pUC19L1-E7,制备HPV16L1-E7重组腺病毒rAd5HPV16L1-E7,HPV16L1-E7融合蛋白可在293细胞中高效表达,可达细胞总蛋白的10％以上,并装配成嵌合病毒样颗粒(cVLP)。结论成功制备了可高效表达IPV16L1-E7嵌合L1-E7VLP的重组腺病毒rAd5HPV16L1-E7载体疫苗,为HPV重组腺病毒疫苗用于宫颈癌的防治打下了基础。构建的pUC19L1载体可以比较方便的插入外源基因,用来构建多价疫苗,因此在制备HPV16多价疫苗方面具有广泛的应用价值。     

Chimeric virus-like particles of the human papillomavirus type 16 (HPV 16) as a prophylactic and therapeutic vaccine

Infection by certain human papillomaviruses (HPV), most notably HPV types 16 and 18, is the major risk factor for cervical cancer. Worldwide, this disease represents the second most frequent malignant tumor in women; thus, there is urgent need for efficient therapy and prevention. The natural history of cervical cancer and its precursors (cervical intraepithelial neoplasias), as well as animal experiments, strongly suggest that the immune system controls both the primary infection (by neutralizing antibodies directed against the major structural protein L1) and the progression of the disease (via cytotoxic T cells specific for the viral oncoproteins expressed in transformed cells, e.g., E7). By the expression of an HPV 16 L1E7 fusion protein, we have generated chimeric virus-like particles (CVLP). Immunization of mice with CVLPs induces neutralizing antibodies directed against L1 virus-like particles (devoid of the E7 portion) and E7-specific T cells as measured in vitro. Vaccinated animals are protected against tumor growth following inoculation of syngeneic HPV 16-transformed cells. In addition, we observed a therapeutic effect of vaccination on pre-existing tumors. This data allowed us to conclude that CVLPs are suitable for prevention and therapy of HPV infection. A vaccine based on HPV 16 L1E7 CVLPs is currently under development.     

大肠癌新型CEA疫苗重组杆状病毒的表达与鉴定

目的利用Bac-to-Bac杆状病毒表达系统,构建新型CEA融合表位疫苗(以HPV16L1为蛋白载体)的重组杆状病毒.方法构建含有HPV16L1片段和CEA-PADRE合成片段的表达载体pFastBacl-Ⅴ,转化DH1OBac感受态菌.利用其含有的细菌Tn7转座系统,将该基因重组至杆状病毒穿梭质粒bacmid中,转染昆虫细胞sf9,获得新型CEA疫苗的重组杆状病毒.结果限制性内切酶酶切和DNA序列分析表明目的片断正确克隆到杆状病毒转移载体pFastBac1中;重组杆状病毒感染昆虫细胞后,PCR检测可以扩增出相应大小的片段.结论利用杆状病毒表达系统,成功构建了CEA融合表位疫苗的重组杆状病毒,为进一步的研究奠定了基础.     

利用Sm抗原模拟表位肽构建狼疮样鼠模型

目的利用Sm抗原模拟表位肽免疫BALB/C小鼠,构建狼疮样鼠模型。方法用鼠Sm单克隆抗体对噬菌体随机12肽库进行亲和筛选,将筛选的噬菌体阳性克隆行双抗体夹心ELISA检测、DNA序列测定并推导其氨基酸序列,初步确定Sm抗原模拟表位;进而用混合噬菌体阳性克隆皮下免疫BALB/C小鼠,ELISA法检测小鼠血清IgG型抗Sm抗体、抗双链DNA（dsDNA）抗体和抗核抗体（ANA）水平;直接免疫荧光检测小鼠肾脏IgG型免疫复合物的沉积以及HE染色观察小鼠肾脏组织病理损伤情况。结果免疫筛选获得的5个ELISA强阳性的噬菌体克隆的氨基酸序列IR、SQ、PP出现频率增加;实验组小鼠血清中第28天出现Sm抗体、dsDNA抗体、ANA抗体,且滴度不断升高;第33天出现蛋白尿;肾脏可以检测到IgG型免疫复合物沉积及明显的肾脏病理改变。对照组小鼠无明显自身抗体产生和肾脏病变。结论利用Sm抗原噬菌体模拟表位肽可成功构建狼疮样小鼠模型。