HPV16L1／L2蛋白单克隆抗体的制备

用真核表达的ＨＰＶ１６Ｌ１／Ｌ２蛋白作为抗原，经免疫、融合、选择性培养、克隆化等过程，我们建立了两株抗ＨＰＶ１６Ｌ１／Ｌ２蛋白的杂交瘤细胞株（另有数株仍在建株中）。免疫斑点法检测证明，它们产生的抗体，只与ＨＰＶ１６Ｌ１／Ｌ２蛋白起反应，而不与ＨＰＶ１６Ｅ６、Ｅ７蛋白、人血清蛋白和牛血清蛋白起反应。另外，免疫组化试验证明，这种抗体可应用于临床ＨＰＶ１６感染的检验，具有敏感、特异、准确、快捷、经济的优点。     

悬浮培养昆虫细胞表达重组HPV16 L1蛋白

目的 用悬浮培养昆虫细胞方式表达人乳头瘤病毒16型(HPV16)L1蛋白,为获得病毒样颗粒(VLPs)及进一步深入研究疫苗和诊断试剂盒打基础.方法 优化昆虫细胞Sf9的悬浮培养条件;优化扩增病毒和蛋白的条件;空斑试验测定病毒滴度;SDS-PAGE和Western Blot分析目的蛋白的表达情况;透射电镜观察细胞内HPV16 L1蛋白形成的VLPs.结果 优化后,悬浮培养昆虫细胞初始接种密度为5×105cell/ml,以MOI(Multiplicity ofInfection)=10接种重组病毒rBacV/HPV16 L1,72～84h收获细胞沉淀为最佳.经透射电镜观察,在重组病毒感染的昆虫细胞内,存在HPV16L1蛋白形成的VLPs.结论 优化了细胞悬浮培养、病毒扩增和蛋白表达的条件;电镜观察在重组病毒rBacV/HPV16L1感染的昆虫细胞中,HPV16 L1蛋白可形成VLPs.     

YY1蛋白对HPV16启动子P97的抑制效应依赖于上游的增强子序列

目的　观测位于HPV16LCR序列YY1结合位点上游的组织特异性增强子序列对YY1蛋白的启动子P97抑制作用的影响。方法　构建带有不同长度的HPV16野生株、启动子远端YY1位点突变株、启动子近端YY1位点突变株的 5′端LCR缺损序列的荧光素酶报导质粒 ,以及不同长度的近端YY1/SP1重叠结合位点基因工程突变LCR的荧光素酶报导质粒 ;将各种质粒转染到人类子宫颈癌细胞系C33A中 ,检测在不同LCR序列的控制下对病毒启动子P97的活性的影响。结果　去除上游增强子序列后 ,远端YY1位点突变的P97激活作用完全消失 ,近端YY1位点突变的P97激活作用部分丧失。不同长度的近端YY1/SP1重叠位点基因工程突变LCR对P97活性的影响不受上游启动子序列的控制。结论　YY1蛋白对启动子P97的抑制效应受位于其上游序列的增强子的控制     

HPV16,HPV18E6单抗SP法染色体会

ＨＰＶ１６、ＨＰＶ１８Ｅ６单抗ＳＰ法染色体会任占平作者单位：广西柳州市第一人民医院病理科５４５００１近年来，人乳头瘤病毒（ＨＰＶ）１６、１８感染已被证实与人宫颈癌、肺癌、食管癌、大肠癌等恶性肿瘤病因学密切相关〔１，２〕。但目前国内对ＨＰＶ１６、ＨＰＶ...     

HPV阳性宫颈细胞学标本及活检组织中L1蛋白表达及意义

目的 探讨HPV L1衣壳蛋白在HPV阳性的宫颈细胞学标本和活检组织标本中的表达及意义.方法 运用免疫组化SP法检测L1蛋白在宫颈细胞学和组织学标本中表达.结果 52例细胞学标本中,L1蛋白在非典型鳞状细胞(ASCUS)、低级别鳞状上皮内病变(LSIL)、高级别鳞状上皮内病变(HSIL)中阳性表达率分别为43.8%(7/16)、28.5%(10/26)、0(0/10);100例组织学标本中,L1蛋白在炎症、CIN1、CIN2、CIN3中阳性表达率分别为55.6%(15/27)、43.8%(14/32)、15.4%(4/26)、0(0/15);两组资料L1蛋白阳性表达率的差异均具有统计学意义(χ2=9.149,P=0.01;χ2=23.555,P=0.00).结论 早期检测HPV L1蛋白,有助于判断宫颈病变程度及其恶性进展趋势,为宫颈病变患者提供更合理的治疗流程.     

原核高效制备人乳头瘤病毒16型L1病毒样颗粒

目的通过原核表达系统高效制备人乳头瘤病毒(HPV)16晚期蛋白L1病毒样颗粒(VLPs)。方法构建HPV16L1基因序列优化前后的PET30aHPV16L1重组质粒,转化大肠杆菌Rosetta(DE3);用IPTG诱导目的基因表达,两步层析方法纯化HPV16L1蛋白;电镜下观察纯化产物形成VLPs的情况。结果成功构建大肠杆菌工程菌,高效可溶表达(目的蛋白约占总蛋白的38%)并纯化HPV16L1蛋白,纯度达95%以上,电镜下观察,发现纯化后的目的蛋白为直径50 nm左右,形态与天然病毒颗粒高度相似。结论在大肠杆菌原核系统中高效、简易地制备了HPV16L1VLPs,为诊断试剂和疫苗的研制奠定基础。     

子宫颈病变中HPV L1蛋白和p16的表达

目的 研究HPV L1蛋白和p16在子宫颈各种病变中的表达情况,探讨它们在子宫颈病变进展中的预测价值.方法 应用免疫组化方法检测41例各种子宫颈病变(CIN1级18例、CIN2级9例、CIN3级8例和浸润性鳞状细胞癌6例)中HPV L1蛋白和p16的表达.结果 HPV L1蛋白在各种子宫颈病变中的阳性率为26.8%.其中HPV L1在CIN1中的阳性表达率为38.9%,CIN2为44.4%,CIN3和浸润性鳞状细胞癌均无表达.p16在各种子宫颈病变中的阳性率为68.3%,其在CIN1中的阳性表达率为38.9%,CIN2为77.8%,CIN3和浸润性鳞状细胞癌均表达阳性.100%CIN3和浸润性鳞状细胞癌为p16+/HPV L1-,而61.1% CIN1中为p16-/HPV L1+或p16-/HPV L1-.结论 随着子宫颈病变的进展,HPV L1阳性表达率降低而p16阳性表达率增高.p16+/HPV L1-提示子宫颈鳞状上皮内瘤变有进展的可能,而p16-/HPV L1+和p16-/HPV L1-可能为无进展的或潜在消退的子宫颈病变.     

宫颈癌组织中人乳头瘤病毒16型L1基因的克隆及序列分析

目的人乳头瘤病毒１６型（ＨＰＶ１６）晚期基因区（Ｌ１）编码病毒的主要衣壳蛋白,且ＨＰＶ１６感染与宫颈癌发生、发展关系密切,故对中国妇女感染的ＨＰＶ１６基因进行克隆及序列分析有重要实际意义。方法采用聚合酶链反应技术（ＰＣＲ）扩增了３例中国妇女宫颈癌组织中ＨＰＶ１６的Ｌ１基因片段,并对其进行了克隆及全序列分析。结果发现３个ＨＰＶ１６Ｌ１基因核苷酸序列有４处均与最初报道的ＨＰＶ１６Ｌ１ＤＮＡ序列不同,并由此引起所编码的氨基酸亦发生变化。结论中国妇女宫颈癌组织中ＨＰＶ１６Ｌ１核苷酸有一定程度变异。     

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。     

YYI蛋白对HPV16启动子P97的抑制效应依赖于上游？…

目的 观测位于ＨＰＶ１６ＬＣＲ序列ＹＹ１结合位点上游的组织特异性增强子序列对ＹＹ１蛋白的启动子Ｐ９７抑制作用的影响。方法 构建带有不同长度的ＨＰＶ１６野生株、启动子远端ＹＹ１位点突变株、启动子近端ＹＹ１位点突变株的５’端ＬＣＲ缺损序列的荧光素酶报导质粒，以及不同长度的近端ＹＹ１／ＳＰ１重叠结合位点基因工程突变ＬＣＲ的荧光素酶报导质粒；将各种质粒转染到人类子宫颈癌细胞系Ｃ３３Ａ中，检测在不同ＬＣＲ序     

Expression of HPV L1 capsid protein in cervical specimens with HPV infection

We tried to investigate the expression rate of human papillomavirus (HPV) L1 capsid protein in uterine cervical specimens and correlate it with the grade of dysplasia, HPV genotype and age of the patients. Among uterine cervical specimens proved to have HPV by DNA genotyping test, eighty cytology-biopsy matched cases and 22 unmatched cytology specimens were selected. Immunostaining for L1 capsid protein was performed on both cervical smears and tissue sections. The L1 capsid protein was expressed mainly in the nuclei, but occasionally in the cytoplasm of cells located in the superficial layer of squamous epithelium. The immunostaining for L1 capsid protein showed positive reaction in 47 cases (46.1%) of cervical smears and in 10 cases (12.5%) of tissue sections (P = 0.001). Cytologic diagnosis revealed a higher expression rate in LSILs (25/33; 75.8%) than in HSILs and cervical cancers (8/20; 40.0% and 2/5; 40%, respectively) (P = 0.006). In LSILs, cases with low-risk type HPV showed a higher L1 capsid expression rate than those with the high-risk type HPV (88.9% vs. 70.8%). The L1 capsid expression rate decreased in the over-40-year-old age group compared to the younger age (49.2% vs. 50.8%). Cytology smears were superior to tissue sections for the detection of L1 capsid protein expression. LSILs and HPV low-risk group showed higher L1 capsid expression rate than HSILs and HPV high-risk group, which suggests that L1 capsid expression might be related to a favorable disease biology.     

An analysis on the combination expression of HPV L1 capsid protein and p16INK4a in cervical lesions

Human papillomavirus (HPV) infection in cervix is the most important reason for cervical cancer, but only 2% cervical HPV infection will develop into cervical cancer. So how to identify patients at risk of progressive cervical lesions from those infected with HPV to avoid over treatment is a big issue in clinic. The aims of this study were to detect the expression of HPV L1 capsid protein and p16^INK4a in cervical lesions and to investigate the combination expression of HPV L1 capsid protein and p16^INK4a in cervical lesions and its diagnostic efficiency in clinic. Immunochemical method was used to detect the expression of HPV L1 capsid protein and p16^INK4a in 169 cases of abnormal cytology. Histopathologic test was performed to identify cervical lesions of all the cases. χ² test and spearman's rank correlation were used for statistical analysis. The diagnostic sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), accuracy, and the area under the receive operating characteristic (ROC) curve (denoted by A_Z) were calculated with SPSS 13.0. All the statistical tests were two sided at the 5% level of significance. L1 expression decreased (P < 0.001), but p16^INK4a expression increased (P < 0.001) with histopathologic diagnosis increasing. The expression rates of HPV L1 capsid protein, p16^INK4a, and L1(?)/p16(+) in cervical intraepithelial neoplasia (CIN)2, CIN3, and squamous‐cell carcinoma were statistically different from those in CIN1 (P < 0.001). The expressions of HPV L1 capsid protein, L1(+)/p16(+), L1(+)/p16(?), and L1(?)/p16(?) were negatively correlated with the severity of cervical lesions (P < 0.001), whereas the expressions of p16^INK4a and L1(?)/p16(+) were positively correlated with the severity of cervical lesions (P < 0.001). The specificity and A_Z of combining L1 with p16 ^INK4a were statistically higher than L1 or p16 ^INK4a alone (P < 0.05). L1 and p16^INK4a are useful biomarkers for the early diagnosis of cervical lesions. The combination of L1 and p16^INK4a has a higher diagnostic accuracy than L1 or p16^INK4a alone in diagnosis of cervical lesions. Diagn. Cytopathol. 2010;38:573–578. © 2009 Wiley‐Liss, Inc.     

Immunohistological analysis of HPV L1 capsid protein and p16 protein in low-grade dysplastic lesions of the uterine cervix

Human papillomavirus (HPV) L1 capsid protein (L1) is associated with the productive phase of HPV infection. However, the expression of L1 and its relationship to p16 expression, a surrogate marker for HPV infection, are unknown. We examined the relationship between L1 and p16 expression in cervical intraepithelial neoplasia. Tissues were divided into four categories: regressive lesions (n=48), progressive lesions (n=40), and randomly selected CIN1-2 (n=67) and CIN3 (n=44). P16 positivity in the progression cases was significantly higher than that in the regression cases, and p16 positivity in the CIN3 cases was significantly higher than that in any other categories. L1 positivity was not significant between each category. The staining pattern was divided into the following four groups: L1-/p16-, L1+/p16-, L1+/p16+, L1-/p16+. The L1-/p16- pattern was significantly associated with the regression nature in CIN1-2. Some CIN3 cases showing a feature of L1+/p16+, which are still HPV-productive, are likely to exist. The combination of both L1 and p16 may be useful in the evaluation of the progression risk of low-grade cervical dysplasia.     

41751例女性宫颈细胞中HPV基因分型的回顾性分析

目的：分析女性宫颈上皮细胞人乳头状瘤病毒HPV感染状况和基因分布情况,为预防HPV感染和防治宫颈癌提供科学依据。方法：收集2013年1月至2015年8月中国人民解放军北京军区总医院宫颈癌筛查自愿接受宫颈HPV检查者宫颈标本41751例,年龄为14~95岁。采用凯普的导流杂交方法对宫颈上皮细胞标本进行15种高危HPV和6种低危HPV基因型检测,分析女性宫颈HPV感染状况。结果：41751例女性宫颈上皮细胞标本HPV总阳性率为17.485%,高危HPV型别前4位的分别是16、52、53和58,低危HPV型别前4位的分别是6、11、42和43。结论：凯普的导流杂交膜HPV各基因亚型位点分布对女性宫颈HPV感染分子流行病学调查研究具有重要意义。     

Clinicopathological Implications of Human Papilloma Virus (HPV) L1 Capsid Protein Immunoreactivity in HPV16-Positive Cervical Cytology

Background: The objective of this study was to investigate the expression of human papilloma virus (HPV) L1 capsid protein in abnormal cervical cytology with HPV16 infection and analyze its association with cervical histopathology in Korean women.Material and Methods: We performed immunocytochemistry for HPV L1 in 475 abnormal cervical cytology samples from patients with HPV16 infections using the Cytoactiv^® HPV L1 screening set. We investigated the expression of HPV L1 in cervical cytology samples and compared it with the results of histopathological examination of surgical specimens.Results: Of a total of 475 cases, 188 (39.6%) were immunocytochemically positive and 287 (60.4%) negative for HPV L1. The immunocytochemical expression rates of HPV L1 in atypical squamous cells of unknown significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and cancer were 21.8%, 59.7%, 19.1%, and 0.0%, respectively. LSIL exhibited the highest rate of HPV L1 positivity. Of a total of 475 cases, the multiple-type HPV infection rate, including HPV16, in HPV L1-negative cytology samples was 27.5%, which was significantly higher than that in HPV L1-positive cytology samples (p = 0.037). The absence of HPV L1 expression in ASCUS and LSIL was significantly associated with high-grade (≥cervical intraepithelial neoplasia [CIN] 2) than low-grade (≤CIN1) histopathology diagnoses (p < 0.05), but was not significantly different between HPV16 single and multiple-type HPV infections (p > 0.05). On the other hand, among 188 HPV L1-positive cases, 30.6% of multiple-type HPV infections showed high-grade histopathology diagnoses (≥CIN3), significantly higher than the percentage of HPV16 single infections (8.6%) (p = 0.0004)Conclusions: Our study demonstrates that the expression of HPV L1 is low in advanced dysplasia. Furthermore, the absence of HPV L1 in HPV16-positive low-grade cytology (i.e., ASCUS and LSIL) is strongly associated with high-grade histopathology diagnoses. The multiplicity of HPV infections may have an important role in high-grade histopathology diagnoses (≥CIN3) in HPV L1-positive cases.     

Detection of HPV 16 and HPV 18 DNA in the blood of patients with cervical cancer

Persistent infection of the uterine cervix with high-risk human papillomaviruses (HPV) is causally associated with cancer of the cervix. A few studies have reported the presence of HPV DNA in the blood of women with cervical neoplasia. The aim of this study was to determine if HPV DNA could be detected in whole blood of women with a range of cervical pathologies and with HPV 16 or 18 cervical infections and if there is a correlation between cervical lesion grade and the appearance of HPV DNA in the circulatory system. Forty-five women with histologically graded cervical cancer were confirmed to have cervical HPV 16 or 18 infections. Eleven (24.4%) of these women had detectable HPV 16 or 18 in their blood. The HPV types detected in the blood matched those detected at the cervix. No HPV 16 or 18 DNA was detected in the blood of 32 women with pre-cursor cervical lesions or normal cervical pathology but who had cervical HPV 16 or 18 infections. One of 77 women with normal cervical pathology and no cervical HPV infection was positive for HPV 16 DNA in her blood. The results indicate that HPV DNA can be detected in the blood of women with more advanced cervical carcinomas but not in the blood of women with pre-cursor cervical lesions. The results of our study indicate that the role of HPV DNA in the circulatory system appears not be of diagnostic significance and HPV DNA is only detectable in women with more advanced cervical cancers.     

Determination of HPV type 16 and 18 viral load in cervical smears of women referred to colposcopy

It has been recognized that human papillomavirus infection is the major causal factor for high-grade cervical lesions. The aim of the study was to evaluate the relationship between HPV 16 and 18 viral loads and cervical status in different age strata. A duplex real time PCR method was devised to determine HPV 16 and 18 viral load per million of human cells using an in house plasmidic construct as a standard of quantification. The 151 cervical scrapes were collected before colposcopic examination from either abnormal cervico-vaginal smear (group 1, 97 patients) or from post treatment clinical follow-up (group 2, 54 patients). In women aged 30-40, the HPV16 viral loads were significantly higher in high-grade squamous intraepithelial lesion than in low-grade squamous intraepithelial lesion in both groups and HPV18 in group 1. In women aged 20-30 of group 1, high HPV viral load was associated in few cases with high-grade squamous intraepithelial lesion or low-grade squamous intraepithelial lesion, and surprisingly in some patients with normal cervix. HPV 16 and 18 viral loads are related to the severity of cervical lesion, and may be useful in the clinical management of cervical lesions. A specific follow-up may be useful for those with high viral load despite normal cervix.