HPV16 L1基因的原核表达及免疫反应性鉴定

目的：在大肠杆菌中表达人乳头瘤病毒16型(HPV16)主要衣壳蛋白L1，并鉴定其免疫反应性。方法：将HPV-16L1基因克隆人原核表达载体pThioHisC中，构建重组表达载体。以重组载体分别转化大肠杆菌Top10和DH5α，在IPTG诱导下表达外源基因，用SDS—PAGE和Western blot对表达产物进行鉴定和分析。结果：构建了HPV—16L1基因的原核表达质粒pThioHisC／HPV—16L1，并在大肠杆菌中表达出相对分子质量(Mr)约为70800的蛋白。表达的蛋白能与抗HPV—16L1抗体发生特异性反应。结论：在原核细胞中成功地表达HPV—16L1基因，为HPV—16L1疫苗的研制提供了必要的基础。     

人乳头瘤病毒16型E4蛋白表达、纯化及抗血清制备

目的 表达人乳头瘤病毒16型(HPV16) E4蛋白,并制备小鼠抗HPV16 E4血清.方法 将HPV16 E4基因克隆入pQE30,重组质粒pQE30-HPV 16E4经鉴定后转化大肠埃希菌M15(PREP4),诱导表达并鉴定表达产物.因纯化、变性和复性方法,制备可溶性HPV16 E4蛋白.免疫Bal B/C小鼠制备抗血清,检测小鼠IFN-γ水平、CD4/CD8比值和抗血清滴度变化.结果 酶切和测序结果表明pQE30-HPV16F4构建成功.表达分子相对分子量(Mr)为10 000,Western blot法证明具有较高特异性.小鼠抗血清效价升高,CD4/CD8无升高,小鼠γ干扰素(IFN-γ)无升高.结论 成功制备可溶性HPV16 E4蛋白和小鼠抗HPV16 E4高效价的抗血清.     

新疆南部地区维吾尔族妇女宫颈癌组织中HPV-16型L2基因多态性分析

目的探讨新疆南部地区维吾尔族妇女宫颈癌组织中人乳头瘤病毒16型(humanpapillomavirus16,HPV16)L2基因的变异,并预测L2蛋白的功能变化。方法从19份中国新疆南部地区维吾尔族妇女宫颈癌活检组织标本中提取DNA,以此DNA为模板,PCR扩增HPV16L2全长基因,PCR产物直接测序或克隆后测序,分析新疆维吾尔族妇女宫颈癌组织HPV16L2基因多态性及HPV16L2蛋白功能的变化。结果PCR检测结果显示宫颈癌组织中HPV16L2阳性率为84.21%(1619);测序和序列分析表明L2基因核苷酸多处发生变异,并引起编码氨基酸的变异;L2基因在核苷酸水平上形成7种突变模式(XJL21～XJL27),各模式与HPV16原型比较,同源性在99.37%～99.79%之间;在氨基酸水平上形成5种突变模式,其中XJL1123突变模式占66.67%(812),是突变的主流模式,各模式与HPV16原型比较,同源性在98.31%～99.58%之间;以上突变引起HPV16L2蛋白疏水性和抗原性的改变,继而改变了L1蛋白的结构及功能。结论中国新疆南部地区维吾尔族妇女宫颈癌组织中HPV16L2基因发生多位点变异,并形成多种突变模式和突变主流模式;这些突变引起HPV16L2蛋白疏水性和抗原性的改变,提示HPV16L2基因突变可能与HPV16的系统发生以及病毒逃避机体免疫识别有关。     

HPV16型E7重组蛋白的表达及其多克隆抗体的制备

目的制备人乳头瘤病毒(HPV)16型E7原核表达蛋白及其多克隆抗体。方法用PCR方法从宫颈癌组织中扩增HPV16 E7基因,克隆至pET21a(+)载体并构建pET21a(+)/HPV16 E7重组质粒,测序鉴定;将重组质粒转化至大肠杆菌BL21(DE3),经异丙基硫代-β-D-硫代吡喃半乳糖苷(IPTG)诱导后表达重组蛋白,用Ni-NTA亲和层析法纯化并经SDS-PAGE及Western blot分析鉴定;纯化的HPV16 E7重组蛋白免疫日本大耳白兔制备多克隆抗体,用ELISA检测多克隆抗体的效价,并用Western blot法及免疫荧光技术分析多克隆抗体的特异性。结果 HPV16 E7重组蛋白可通过原核表达系统进行表达和纯化;通过兔免疫后可制备特异性的多克隆IgG抗体,效价达1∶30 000;经Western blot法和免疫荧光染色结果证实兔多克隆抗体可特异性识别HPV16 E7蛋白。结论成功进行了HPV16 E7原核表达并制备了HPV16 E7兔多克隆抗体。     

HPVL1保守序列肽段诱导多型别HPV抗体的初步研究

为探索一段人类乳头瘤病毒(HPV)L1蛋白中长12个氨基酸残基的保守序列是否能诱导产生多型别HPV抗体,我们通过B细胞表位预测和多序列对比,筛选出一条HPV L1蛋白保守肽段,并人工合成此肽段,加弗氏佐剂后免疫家兔,对照组只用弗氏佐剂。先用ELISA的方法检测免疫家兔血清中抗体滴度;再用免疫细胞化学、免疫细胞荧光、Western blot和免疫组织化学的方法检测此抗血清与HPV阳性的宫颈癌细胞株和宫颈组织的反应情况。结果发现,用ELISA法检测抗血清效价在1∶25600以上,而免疫细胞化学、细胞免疫荧光、Western blot和免疫组织化学等方法均检测出抗血清能与多型别HPV进行反应,而对照组呈阴性。此结果表明,该多肽可诱导产生针对多型别HPV的抗体,对于研发制备HPV、HPV L1诊断试剂盒有重要意义。     

HPV L1 C-末端保守序列短肽诱导HPV多型别交叉抗体的初步研究

通过对HPV L1序列进行比对,发现HPV L1 C-末端存在长30个氨基酸残基的保守序列短肽;出于检测短肽是否可以诱生HPV多种型别交叉抗体的目的,将该序列短肽加弗氏佐剂用于日本大耳白兔和BALB/c小鼠免疫,然后用ELISA方法检测此免疫动物血清及其分泌物中的IgG抗体滴度,发现此免疫动物体内已诱生出高滴度的血清IgG抗体(>1∶20000);再用ELISA、免疫组织化学和Western blot的方法对此诱生血清抗体与HPV阳性宫颈癌细胞株的反应情况进行检测,发现这些短肽抗血清可与16、18型HPV L1很好地进行反应,其对照组呈现阴性。这一研究结果表明短肽可以诱生HPV多种型别交叉抗体。它对后续研发HPV L1广谱疫苗或检测试剂盒具有重要意义。     

人乳头瘤病毒16型L1-E7c嵌合基因的构建表达及免疫学效应的研究

目的 构建人乳头瘤病毒16型(HPV16)L1-ETc嵌合基因并表达融合蛋白,以期获得防治HPV16感染及相关肿瘤的疫苗.方法 以HPV16型的中国人野毒株为模板,利用PCR克隆技术制备HPV16 L1-E7c嵌合基因,并转入原核表达载体pET28a( ),获得pET28a( )-L1-E7c表达质粒.以Western blot方法鉴定融合蛋白与HPV16 L1和E7抗体的特异性结合.应用蛋白纯化仪纯化HPV16 L1-E7c融合蛋白,经过复性后,电镜观察病毒样颗粒(cVLP)的形态结构.纯化的蛋白免疫动物,测定疫苗抗肿瘤的细胞免疫及抗体产生情况.结果 经过序列分析和酶切鉴定表明成功构建了HPV16L1-E7c嵌合基因,并在大肠杆菌中可高效表达L1-E7c融合蛋白.此融合蛋白具有HPV16 L1和E7的抗原性,纯化的蛋白复性后可形成嵌合病毒样颗粒(cVLP),纯化的蛋白免疫动物后,产生特异性细胞和体液免疫反应,具有抗肿瘤活性.结论 构建的嵌合基因HPV16L1-E7c可有效表达HPV16 L1-E7c重组蛋白并形成cVLP.此蛋白在动物实验中具有疫苗的免疫保护作用,为HPV16疫苗的研究奠定了实验基础.     

HPV16型E6重组蛋白的表达及其兔多克隆抗体的制备

目的制备人乳头瘤病毒(HPV)16型E6原核表达蛋白及其多克隆抗体。方法通过PCR法从Siha细胞株中获取HPV16 E6基因后克隆至pET21a(+)载体,构建pET21a(+)/HPV16 E6重组质粒,并经测序鉴定;将重组质粒pET21a(+)/HPV16 E6转化至E.coli BL21(DE3)中,通过异丙基硫代-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达HPV16 E6-His标签融合蛋白,经镍柱亲和层析纯化,然后通过Western blot法鉴定;将纯化的HPV16型E6重组蛋白免疫日本大耳白兔制备多克隆抗体, ELISA检测免疫后血清多克隆抗体效价,并用Western blot法和免疫荧光技术分析HPV16 E6兔多克隆抗体的特异性。结果成功构建pET21a(+)/HPV16 E6重组质粒,并经测序鉴定;在重组质粒pET21a(+)/HPV16 E6转化至大肠杆菌BL21(DE3)后,经亲和层析法获得纯化的HPV16型E6重组蛋白,用于免疫日本大耳白兔获得多克隆抗体,抗体效价为1∶40 000,并用Western blot法和免疫荧光技术确认了多克隆抗体的特异性。结论 HPV16 E6原核表达成功并制备了HPV16 E6兔多克隆抗体。     

HPV31和52 L2融合蛋白的原核高效表达及免疫效果评价

目的 高效表达HPV31和52型L2融合蛋白疫苗,并评价其免疫效果.方法 根据GenBank上公布的HPV31、52型L2蛋白11-200位氨基酸序列,设计合成两个型别L2该区域对应的优化密码子基因融合序列,并将其克隆至原核表达载体中.利用原核表达系统表达HPV31和52 L2融合蛋白,纯化目的蛋白后免疫小鼠,用血清抗体检测及HPV假病毒体外中和试验评价融合蛋白疫苗的免疫效果.结果 HPV31和52 L2融合蛋白在原核表达体系中呈高效表达,表达量约占全菌20％.融合蛋白加铝佐剂免疫小鼠后,血清中能检测到高滴度的总抗体及一定水平的中和抗体和交叉中和抗体.结论 HPV31和52 L2融合蛋白能够刺激机体产生广谱中和抗体,为高危型广谱HPVL2蛋白疫苗研发提供了实验室依据.     

Expression of the human papillomavirus type 6b L2 open reading frame in Escherichia coli: L2-beta-galactosidase fusion proteins and their antigenic properties

Human papillomavirus (HPV) type 6b genome contains two large open reading frames (ORFs), designated L1 and L2, in a putative late region. These ORFs are expected to code for viral structural proteins. To examine antigenic properties of a L2 gene product, we constructed two plasmids which contain N-terminal (L2-N) and internal (L2-I) regions of the HPV6b L2 ORF and then each region was expressed in Escherichia coli as a fusion protein with E. coli beta-galactosidase (beta-Gal). Both L2-N/beta-Gal fusion proteins reacted with anti-beta-Gal antibody, but did not react with the antibody prepared against bovine papillomavirus type 1 (BPV1), in contrast with a high reactivity of HPV6b L1-beta-Gal fusion protein with the anti-BPV1 antibody. Antibody raised against the L2-I/beta-Gal protein in a rabbit reacted with viral antigens in the nuclei of cells in superficial epithelium of the condyloma acuminatum tissue, but did not react with the antigens in the bovine papilloma tissue. This antibody recognized a protein from condyloma acuminata which migrates to the position of mol wt 70K-76K on an electrophoresed SDS-polyacrylamide gel. These results suggested that the L2 ORF of HPV6b codes for a capsid protein which is less cross-reactive than the L1 antigen with anti-BPV1 antibody.     

A comparison of the detection of biomarkers in infections due to low risk versus high-risk human papillomavirus types

Adjunctive immunohistochemistry tests for human papillomavirus (HPV) infection include p16 and Ki67 as well as the more recently discovered biomarkers importin-β, exportin-5, Mcl1, and PDL1. The purpose of this study was to compare the expression of these biomarkers in HPV infection due to the high-risk types such as HPVs 16, 18, 31, 33, 35, and 51 versus lesions that contain the low risk types HPV 2, 6 or 11. We studied 35 lesions with low risk HPV types (verruca vulgaris = 10 cases, condyloma acuminatum = 15 cases, CIN 1 with HPV 6/11 = 10 cases) and 25 CIN 1 or 2 lesions with a high-risk HPV type. The 25 high-risk positive CIN 1–2 cases had strong expression of the panel p16, Ki67, importin-β, exportin-5, Mcl1, and PDL1 where each protein localized to the cells in the parabasal aspect of the lesion. In comparison, neither p16, importin-β, exportin-5, Mcl1, nor PDL1 were increased in the epithelia of the lesions with the low risk HPV types; Ki67 showed variable expression. HPV viral capsid L1 protein and viral DNA were excellent markers of infection in the lesions with low risk types. Thus, p16, importin-β, exportin-5, Mcl1, and PDL1 are not only biomarkers of high-risk HPV infection but can also differentiate such lesions from those that contain low risk HPV types. Low risk HPV infections can be best differentiated from their mimics by viral L1 capsid detection and/or HPV DNA by in situ hybridization.     

融合基因Tat-HPV16L1的克隆及其在酿酒酵母中的表达

目的构建人乳头瘤病毒16型L1基因和蛋白转导肽的Tat基因的融合表达载体．导入酿酒酵母表达系统进行诱导表达,为口服基因疫苗的制备打下基础。方法以pBR322-HPV16质粒为模板扩增L1基因,与酿酒酵母表达载体pYES2连接,重组质粒再与自行设计的蛋白转导功能片段Tat连接,经测序鉴定后转入酿酒酵母表达菌株INVSc1诱导表达Tat-HPV16L1融合蛋白。结果融合基因Tat—HPV16L1测序结果与预期完全符合,经过半乳糖诱导表达后进行SDS-PAGE电泳和Western blotting分析,结果表明诱导表达成功。结论成功构建和表达了融合表达载体pYES2＋Tat-HPV16L1,为制备以酿酒酵母为运送载体的口服人乳头瘤病毒疫苗打下基础。     

Detection of human papillomavirus types 6 and 11 E4 gene products in condylomata acuminatum.

Polyclonal antiserum to an Escherichia coli-produced beta-galactosidase/E4 fusion protein of human papillomavirus type 6b (antiserum 256), and affinity purified HPV 11 anti-E4 antibodies were tested for reactivity in Western blots with bacterially expressed trpE/E4 fusion proteins of HPV types 6b, 11, 16, and 18. To further characterize the affinity purified anti-E4 antibodies, a dot-immunobinding assay was performed using overlapping synthetic HPV 11 E1E4 peptides as antigens. Protein extracts of condylomata acuminatum from 18 patients containing HPV type 6 or 11 DNA sequences were tested in Western blots using antiserum 256 or affinity purified HPV 11 anti-E4 antibodies. In the Western blots of the trpE proteins, antiserum 256 identified the HPV types 6b and 11 fusion proteins; the affinity purified HPV 11 anti-E4 antibodies identified only the HPV 11 fusion protein. In the dot-immunobinding assay, three HPV 11 peptides were recognized, each containing a shared 8 amino acid sequence that differs significantly from the corresponding sequences of HPV types 6b, 16, or 18. In the Western blots of protein extracts from 18 condylomata acuminatum samples shown to contain HPV types 6 or 11 DNA, putative E4 gene products were identified in six samples by antiserum 256. The affinity purified HPV 11 anti-E4 antibodies identified putative E4 gene products in one of these same six lesions, which was shown to contain HPV 11 sequences by the Southern blot method. All six samples containing E4 gene products were from women. Three of these women were pregnant, one had serum antibodies to the human immunodeficiency virus, and one was a renal transplant recipient receiving glucocorticoids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two newly identified human papillomavirus types (HPV 40 and 57) isolated from mucosal lesions

Two new papillomaviruses, HPV 40 and HPV 57, were isolated from a PIN lesion and an inverted papilloma of the maxillary sinus, respectively. HPV 40 showed a 13% homology to HPV 7 by reassociation kinetics and HPV 57 showed a 17% homology to HPV 2 and 25% homology to HPV 27. Hybridization of the DNA of these papillomaviruses to a wide variety of different tumor biopsies revealed that HPV 40 was present in a few genital condylomata acuminata as well as in bowenoid lesions. HPV 57 DNA was present in an oral wart, a genital condyloma acuminatum, and verrucae vulgares lesions from two immunosuppressed patients.     

人乳头瘤病毒16型L1/E7融合蛋白在原核细胞中的表达及免疫效果观察

目的构建能表达L1E7融合蛋白的原核表达菌株,纯化蛋白,并观察其免疫效果。方法用PCR方法分别扩增出C末端部分缺失的HPV16L1基因和HPV16E7编码基因N端部分序列。将上述基因连接,构建融合基因L1ΔCE7N并将其插到原核表达载体pGEX-2T中进行融合蛋白表达纯化,然后观察其免疫效果。结果L1ΔCE7N融合基因测序结果表明,序列与设计相符,读码框架正确。将其插入原核表达质粒在大肠埃希菌中获得高效表达;经Wester-Blot鉴定在相对分子质量约85×103处有特异性表达带,与预期相符。用亲和层析和分子筛可纯化L1ΔCE7N融合蛋白,将其免疫C57BL/6小鼠,结果表明融合蛋白能诱发高滴度L1、E7抗体,并能保护小鼠免受TC-1肿瘤细胞的攻击。结论本实验在原核系统中高效表达并纯化了L1ΔCE7N融合蛋白,该蛋白可作为预防和治疗HPV16感染以及相关肿瘤的候选疫苗株。为研制HPV16预防治疗性疫苗探索一条经济、易普及的途径。     

Ultrastructural visualization of human papillomavirus DNA in verrucous and precancerous squamous lesions.

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dot-like positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA.     

Ultrastructural Visualization of Human Papillomavirus DNA in Verrucous and Precancerous Squamous Lesions

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dotlike positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA. Acta Pathol Jpn 41: 757-762, 1991.     

Definition of linear antigenic regions of the HPV16 L1 capsid protein using synthetic virion-like particles.

Mice of three haplotypes (H-2d, H-2b, and H-2d/b) were immunized with synthetic HPV16 virus-like particles (VLPs), produced using a vaccinia virus doubly recombinant for the L1 and L2 proteins of HPV16. The resultant anti-VLP antisera recognized HPV16 capsids by ELISA assay and baculovirus recombinant HPV16 L1 and L2 protein on immunoblot. Overlapping peptides corresponding to the HPV16 L1 amino acid sequence were used to define the immunoreactive regions of the L1 protein. The majority of the L1 peptides were reactive with IgG from the mice immunized with the synthetic HPV16 capsids. A computer algorithm predicted seven B epitopes in HPV16 L1, five of which lay within peptides strongly reactive with the murine antisera. The murine anti-VLP antisera failed to react with the two peptides recognized by anti-HPV16L1 monoclonal antibodies raised by others against recombinant L1 fusion protein. We conclude that the immunoreactive epitopes of HPV16 defined using virus-like particles differ significantly from those defined using recombinant HPV16 L1 fusion proteins, which implies that such fusion proteins may not be the antigens to look for HPV16L1 specific immune responses in HPV-infected patients.     

人乳头瘤病毒16L1原核表达质粒的构建

目的：构建人乳头瘤病毒16L1的原核表达质粒,为下一步探讨蛋白的表达以及蛋白的功能研究作准备。方法：以临床HPV16阳性标本为模板,PCR扩增L1的片段,L1片段经EcoRI和SalⅠ双酶切后,插入载体pGEX4T-1,转化JM109感受态细胞,平板筛选获得pGEX4T-1／HPV L1的阳性质粒。通过酶切、测序验证质粒的正确性。结果：构建了原核表达质粒pGEX4T-1／HPV16L1,并通过酶切、测序等方法验证其完全正确。结论：成功构建的pGEX4T-1／HPVL1为今后的蛋白的表达和功能研究打好了坚实的基础,为HPV16的疫苗研究提供了有益的支持。