外源性绵羊肺腺瘤病毒新疆株env囊膜蛋白生物信息学分析

目的采用生物信息学方法分析外源性绵羊肺腺瘤病毒新疆株env囊膜蛋白的结构和功能。方法利用NCBI数据库查询外源性对绵羊肺腺瘤病毒新疆株env囊膜蛋白基因序列,采用在线软件ProtParam、Prot Scale、DNASTAR、SOPMA、SWISS MODEL、SOSUI、TMHMM2.0、Signal 4.1、Bepipred Linear Epitope Prediction、PROSITE SCAN、PSOTR分析预测env蛋白的理化性质、亲疏水性、二级结构、三级结构、跨膜区、信号肽、B细胞表位、结构域、亚细胞定位。结果 env蛋白由615个氨基酸组成,分子式为C_(3144)H_(4924)N_(840)O_(879)S_(29),相对分子质量为69.5×10~3,理论等电点为8.48,不稳定系数为37.04(为稳定蛋白),脂肪系数平均为99.56,总平均亲水性为-0.048。二级结构以α-螺旋为主,占42.76%,无规卷曲占38.7%,延伸链占16.59%,β-转角占1.59%。三级结构env蛋白序列与同源模板的序列在408～540区域存在21%的同源性,只能进行部分同源建模。env蛋白存在2个跨膜区,无信号肽。env蛋白包含有20个线性表位,其中356-442、490-555、104-131、206-230、448-469区段是env蛋白的优势抗原表位区段。env蛋白包含9个N-糖基化位点,4个蛋白激酶C磷酸化作用位点,4个酪蛋白激酶Ⅱ磷酸化作用位点,4个N-豆蔻酰基化位点,1个酰胺化位点,1个细胞附着序列位点。亚细胞定位分析预测env蛋白为细胞膜蛋白,env蛋白绵羊肺腺瘤病毒的囊膜蛋白。结论外源性绵羊肺腺瘤病毒新疆株env囊膜蛋白存在多个B细胞抗原表位及1个细胞附着序列位点,可作为潜在抗原用于env蛋白绵羊肺腺瘤病毒感染疫苗的研制。     

绵羊肺腺瘤病毒的巢式RT—PCR技术检测

目的为能够更加准确、敏感的检测绵羊肺腺瘤病（sheep pulmonary adenomatosis SPA）。方法本试验通过外源性绵羊肺腺瘤病毒的囊膜基因env的TM区和长末端重复序列LTR的U3区分别设计特异性的内外侧引物,运用巢式RT-PCR的方法进行检测,同时与普通RT—PCR进行比较。结果测得的绵羊肺腺瘤病毒的env基因和U3区序列与Gen—Bank中发表的外源性绵羊肺腺瘤病毒基因序列（AF105220）同源性分别为100％和98％。特异性试验结果表明本试验设计的env基因和LTR的U3区的内外侧引物不能从健康绵羊、小鼠、家兔的肺组织以及感染了SPA羊的肾、肝、脾的RNA中扩增出条带。敏感性试验结果表明,env基因和LTR的U3区的巢式RT—PCR诊断方法最低能检测出的标准模板RNA量分别为48fg和48pg,而普通的RT—PCR,env基因及LTR的U3区的最低检出量分别为0．48ng和4．8ng,得出巢式RT—PCR的敏感性高于普通RT—PCR,同时env基因的敏感性高于LTR的U3区的敏感性。结论以上试验说明巢式RT—PCR技术对于检测绵羊肺腺瘤病具有很高的敏感性和实用性。     

在重组痘苗病毒中共表达HIV-1env与hIL-6基因

本实验以 HIV- 1env与 h IL - 6基因在重组痘苗病毒中的共表达研究为目的 ,将痘苗病毒复制非必需区血凝素(HA )基因作为侧翼 ,把编码人白细胞介素 6 (h IL - 6 )的基因片段克隆到真核表达质粒 p J38env的下游 ,构建成含有 HIV- 1env与 h IL - 6两种外源基因的重组表达质粒 PJ38E- IL 6 ,经同源重组和血凝素阴性空斑筛选 ,获得了重组痘苗病毒 v J38E- IL 6。经间接免疫荧光试验、Dot- EL ISA和 Western bolt等检测证明 ,重组病毒能同时表达 env蛋白和 IL - 6蛋白 ,表达产物的分子量分别为 98k Da、2 6 k Da     

绵羊肺腺瘤病毒Gag蛋白的生物信息学分析及抗原表位预测

目的探讨绵羊肺腺瘤病毒(jaagsiekte sheep retrovirus,JSRV)Gag蛋白的组成结构及生物学功能。方法从NCBI数据库中获取JSRV的gag基因序列及其编码蛋白的氨基酸序列,利用ExPASy,ProtScale,Signal 5.0,TMHMM Server,NetPhos Server,NetNGlyc,SOPMA,Prediction,Swiss-Model和DNAStar等生物信息学软件分析Gag的氨基酸序列组成、理化性质、亲疏水性、信号肽、跨膜区、磷酸化位点、糖基化位点、蛋白功能区域、二、三级结构和B、T细胞抗原表位等。结果 Gag蛋白是由611个氨基酸构成富含脯氨酸的亲水蛋白,分子式为C_(3024)H_(4708)N_(834)O_(898)S_(27),相对分子质量为67.981×10~3,理论等电点为7.14;二级结构以无规则卷曲居多,占48.77%;无信号肽且无跨膜区,存在33个磷酸化位点,1个糖基化位点,含有2个超级家族保守结构域和1个锌指结构。预测该蛋白含有17个B细胞优势抗原表位和25个优势CTL表位。结论生物信息学分析JSRV Gag蛋白属于亲水蛋白,含有多个抗原表位,可能具有免疫原性,可作为潜在抗原用于诊断方法的建立与疫苗的研制。     

慢病毒载体介导CD55抑制对胰腺癌BxPC-3细胞迁移、侵袭和成瘤的影响

目的探讨慢病毒载体介导的RNA沉默CD55基因抑制胰腺癌BxPC-3细胞迁移、侵袭和成瘤的效果及机制。方法采用CD55-RNAi-LV(慢病毒包装靶向抑制CD55的shRNA载体)转染BxPC-3细胞(实验组),设转染NC-GFP-LV(慢病毒包装作对照的空载体)的BxPC-3细胞为对照组,未转染的BxPC-3细胞为空白组。Transwell法检测各组细胞体外迁移、侵袭能力。三组细胞分别以1.0×107/200μL的浓度注入裸鼠皮下,7周观察成瘤情况。结果与对照组及空白组比较,实验组细胞迁移能力及侵袭能力明显减弱(P均<0.05);瘤体出现晚且瘤体体积小(P<0.05)。结论 CD55基因对胰腺癌细胞系BxPC-3细胞迁移、侵袭和成瘤能力有调控作用,可望成为胰腺癌新的治疗靶点。     

人乳头状瘤病毒16型E6E7基因与MCA、TPA协同诱导细胞恶性转化的实验研究

目的探讨人乳头状瘤病毒(HPV)16型E6E7基因与化学致癌物MCA、TPA对Balb/c 3T3细胞恶性转化的协同作用。方法构建含HPV16 E6E7基因的重组质粒,用其转染Balb/c 3T3细胞。采用RT-PCR和Westernblot技术检测HPV16 E6E7基因和蛋白表达;应用细胞转化实验研究由MCA和TPA诱导的细胞恶性转化;并检验转化细胞在软琼脂上形成集落的能力及对SCID小鼠的致瘤能力。结果转染HPV16 E6E7基因的细胞比未转染细胞形成更多的转化灶,转化灶个数增加4～25倍,且实验时间明显缩短;其转化细胞在软琼脂上形成集落的能力及对SCID小鼠的致瘤能力更强。结论 HPV16 E6E7基因与MCA、TPA可协同诱导Balb/c 3T3细胞恶性转化。     

人类巨细胞病毒在脐动脉血管平滑肌细胞中的增殖

目的 研究人类巨细胞病毒对血管平滑肌细胞的感染性,为人类巨细胞病毒致动脉粥样硬化及移植物血管病机制研究建立细胞模型.方法 自脐动脉分离血管平滑肌细胞,以1 个感染复数(MOI)的人类巨细胞病毒感染该细胞,观察细胞病变效应,以RT-PCR技术检测人类巨细胞病毒 IE基因在平滑肌细胞内的表达,同时以电镜技术检测平滑肌细胞内的病毒颗粒.结果 人类巨细胞病毒感染脐动脉血管平滑肌细胞后第3天即可出现细胞病变,至第8天已出现明显的细胞病变效应,以RT-PCR技术可从感染人类巨细胞病毒 AD169株的平滑肌细胞内扩增出预期的产物,经测序验证为人类巨细胞病毒 IE基因,以电镜技术可从感染人类巨细胞病毒 AD169株的血管平滑肌细胞中观察到病毒颗粒.结论 人类巨细胞病毒 AD169株可以感染人血管平滑肌细胞,并复制出子代病毒颗粒.为人类巨细胞病毒致动脉粥样硬化及移植物血管病的机制研究提供了较好的实验依据及细胞模型.     

人乳头瘤病毒16型E7疫苗的研究进展

自从 1977年ZurHausen氏提出人乳头瘤病毒 (HuamnPapil lomavirus ,HPV)是妇女宫颈癌病因的假说以来 ,几十年来对HPV的基因及蛋白等结构和功能的研究表明 ,HPV16型是引发宫颈癌等多种肿瘤的主要病毒。它的致癌基因E6/E7在HPV16型致病过程中有重要的作用。针对于HPV16型E7的疫苗研究将有可能为病毒引起的肿瘤的预防和治疗带来新的途径。1　人乳头瘤病毒 16型 (HPV16)与疾病的关系乳头瘤病毒的许多基本特征不同于空泡病毒 ,已经被分成一类独立的病毒[1] 。人乳头瘤病毒以人为单一宿主 ,常存在于人体各部 ,是嗜上皮性病毒。主要引起…     

绵羊肺腺瘤病毒Gag蛋白的生物信息学分析及抗原表位预测北大核心CSCD

目的探讨绵羊肺腺瘤病毒(jaagsiekte sheep retrovirus,JSRV)Gag蛋白的组成结构及生物学功能。方法从NCBI数据库中获取JSRV的gag基因序列及其编码蛋白的氨基酸序列,利用ExPASy,ProtScale,Signal 5.0,TMHMM Server,NetPhos Server,NetNGlyc,SOPMA,Prediction,Swiss-Model和DNAStar等生物信息学软件分析Gag的氨基酸序列组成、理化性质、亲疏水性、信号肽、跨膜区、磷酸化位点、糖基化位点、蛋白功能区域、二、三级结构和B、T细胞抗原表位等。结果Gag蛋白是由611个氨基酸构成富含脯氨酸的亲水蛋白,分子式为C3024H4708N834O898S27,相对分子质量为67.981×103,理论等电点为7.14;二级结构以无规则卷曲居多,占48.77%;无信号肽且无跨膜区,存在33个磷酸化位点,1个糖基化位点,含有2个超级家族保守结构域和1个锌指结构。预测该蛋白含有17个B细胞优势抗原表位和25个优势CTL表位。结论生物信息学分析JSRV Gag蛋白属于亲水蛋白,含有多个抗原表位,可能具有免疫原性,可作为潜在抗原用于诊断方法的建立与疫苗的研制。     

EB病毒感染相关淋巴瘤的研究进展

正EB病毒(Epstein-Barr virus,EBV)是已知的肿瘤相关病毒,其感染与NK/T细胞淋巴瘤、Burkitt淋巴瘤、弥漫大B细胞淋巴瘤及霍奇金淋巴瘤等发生、发展密切相关。而随着检测技术的进步,对EBV致瘤机制的研究也愈加深入。本文就近年来该病毒与相关淋巴瘤的研究进展做如下综述。1 EBV EBV又称人类疱疹病毒4型,属γ-DNA疱疹病毒科。它由Epstein-Barr在1964年发现并命名,当时它的发现者正在研究非洲儿童Burkitt淋     

Jaagsiekte sheep retrovirus biology and oncogenesis

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a lung cancer in sheep known as ovine pulmonary adenocarcinoma (OPA). The disease has been identified around the world in several breeds of sheep and goats, and JSRV infection typically has a serious impact on affected flocks. In addition, studies on OPA are an excellent model for human lung carcinogenesis. A unique feature of JSRV is that its envelope (Env) protein functions as an oncogene. The JSRV Env-induced transformation or oncogenesis has been studied in a variety of cell systems and in animal models. Moreover, JSRV studies have provided insights into retroviral genomic RNA export/expression mechanisms. JSRV encodes a trans-acting factor (Rej) within the env gene necessary for the synthesis of Gag protein from unspliced viral RNA. This review summarizes research pertaining to JSRV-induced pathogenesis, Env transformation, and other aspects of JSRV biology.     

Evidence for a protein related immunologically to the jaagsiekte sheep retrovirus in some human lung tumours.

Human bronchioloalveolar carcinoma (BAC) is a lung cancer, morphologically similar to an endemic contagious lung neoplasm of sheep called sheep pulmonary adenomatosis (SPA) or jaagsiekte. SPA is caused by an exogenous type B/D retrovirus (jaagsiekte sheep retrovirus (JSRV)), which prompted the present study to obtain evidence of a retrovirus in BAC. A panel of 249 human lung tumours, 21 nontumour lung lesions, four normal lung tissues, 23 adenocarcinomas from other organs and a cell line expressing a human endogenous retrovirus protein was examined immunohistochemically using a rabbit antiserum directed against the JSRV capsid protein. Specific staining was detected only in the cytoplasm of recognizably neoplastic cells in the pulmonary alveoli of 39 of 129 (30%) BACs, 17 of 65 (26%) lung adenocarcinomas and two of seven large cell carcinomas. The remaining samples were negative. These results support the hypothesis that some human pulmonary tumours may be associated with a jaagsiekte sheep retrovirus-related retrovirus, warranting further studies.     

Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation

Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope (env) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2/LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for approximately 25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.     

Asthma and Risk of Death from Lung Cancer: NHANES II Mortality Study

Objective. Although smoking is the most important risk factor for lung cancer, nearly 10% of lung cancer is not attributable to smoking. Insights into risk factors for lung cancer other than smoking will become increasingly important, given decreasing trends in the prevalence of smoking. Prior research suggests asthma may increase the risk of lung cancer, particularly among nonsmokers. Methods. We used Cox regression analyses of data from a nationally representative sample of 9087 adults aged 30-75 years included in the NHANES II Mortality Study (1976-1992) to estimate the relative risk (RR) of death from lung cancer associated with self-reported asthma, independent of smoking. Results. Age-adjusted prevalence of smoking was 36.0%, and the age-adjusted prevalence of asthma was 6.1% (6.2% among nonsmokers) at baseline. During approximately 17 years of follow-up, 196 adults died of lung cancer (ICD-9 160-165). Among 6144 nonsmokers, the RR of lung cancer death comparing adults with asthma to those without was 1.69 (95% CI: 0.94-3.04) although the association was not statistically significant. For nonsmokers without a history of cancer, the RR was 2.53 (95% CI: 1.42-4.52). After exclusion of adults with emphysema and chronic bronchitis, the RR of lung cancer death associated with asthma was 3.54 (95% CI: 1.93-6.42). Conclusions. Consistent with prior reports, we observed an increased risk of lung cancer mortality associated with asthma among nonsmokers without a history of cancer.     

Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals.

The jaagsiekte sheep retrovirus (JSRV), which appears to be a type B/D retrovirus chimera, has been incriminated as the cause of ovine pulmonary carcinoma. Recent studies suggest that the sequences related to this virus are found in the genomes of normal sheep and goats. To learn whether there are breeds of sheep that lack the endogenous viral sequences and to study their distribution among other groups of mammals, we surveyed several domestic sheep and goat breeds, other ungulates, and various mammal groups for sequences related to JSRV. Probes prepared from the envelope (SU) region of JSRV and the capsid (CA) region of a Peruvian type D virus related to JSRV were used in Southern blot hybridization with genomic DNA followed by low- and high-stringency washes. Fifteen to 20 CA and SU bands were found in all members of the 13 breeds of domestic sheep and 6 breeds of goats tested. There were similar findings in 6 wild Ovis and Capra genera. Within 22 other genera of Bovidae including domestic cattle, and 7 other families of Artiodactyla including Cervidae, there were usually a few CA or SU bands at low stringency and rare bands at high stringency. Among 16 phylogenetically distant genera, there were generally fewer bands hybridizing with either probe. These results reveal wide-spread phylogenetic distribution of endogenous type B and type D retroviral sequences related to JSRV among mammals and argue for further investigation of their potential role in disease.     

Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary carcinoma, a unique animal model for human bronchioalveolar carcinoma. We previously isolated a JSRV proviral clone and showed that it was both infectious and oncogenic. Thus JSRV is necessary and sufficient for the development of ovine pulmonary carcinoma, but no data are available on the mechanisms of transformation. Inspection of the JSRV genome reveals standard retroviral genes, but no evidence for a viral oncogene. However, an alternate ORF in pol (orf-x) might be a candidate for a transforming gene. We tested whether the JSRV genome might encode a transforming gene by transfecting an expression plasmid for JSRV [pCMVJS21, driven by the cytomegalovirus (CMV) immediate early promoter] into mouse NIH 3T3 cells. Foci of transformed cells appeared in the transfected cultures 2-3 weeks posttransfection; cloned transformants showed anchorage independence for growth, and they expressed JSRV RNA. These results indicate that the JRSV genome contains information with direct transforming potential for NIH 3T3 cells. Transfection of a mutated version of pCMVJS21 in which the orf-x protein was terminated by two stop codons also gave transformed foci. Thus, orf-x was eliminated as the candidate transforming gene. In addition, another derivative of pCMVJS21 (pCMVJS21DeltaGP) in which the gag, pol (and orf-x) coding sequences were deleted also gave transformed foci. These results indicate that the envelope gene carries the transforming potential. This is an unusual example of a native retroviral structural protein with transformation potential.     

Late viral interference induced by transdominant Gag of an endogenous retrovirus

The sheep genome harbors approximately 20 copies of endogenous retroviruses (enJSRVs) closely related to the exogenous and oncogenic Jaagsiekte sheep retrovirus (JSRV). One of the enJSRV loci, enJS56A1, has a defect for viral exit. We report a previously uncharacterized mechanism of retroviral interference. The defect possessed by enJS56A1 is determined by its Gag protein and is transdominant over the exogenous JSRV. By electron microscopy, cells transfected by enJS56A1, with or without JSRV, show agglomerates of tightly packed intracellular particles most abundant in the perinuclear area. The defect in exit and ability to interfere with JSRV exit could be largely attributed to the presence of tryptophan, rather than arginine, at position 21 of enJS56A1 Gag; C98 and V102 also contribute to these properties. We found that enJS56A1 or similar loci containing W21, C98, and V102 are expressed in sheep endometrium. enJS56A1 is a previously unrecognized example of a naturally occurring endogenous retrovirus expressing a dominant negative Gag acting at a late step of the viral replication cycle. Understanding the late blockade exerted by enJS56A1 could unravel fundamental aspects of retroviral biology and help to devise new antiretroviral strategies.     

Small cell lung cancer; recent advances of its biology and therapeutic perspective

Lung cancer is historically divided into two major categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). While the therapeutic efficacy of NSCLC has improved due to the development of molecular targeted therapy and immune checkpoint inhibitors (ICIs) treatment, there has been very slow progress in the therapeutic advances of SCLC. Since SCLC is a deadly disease with rapid progression and early metastasis and comprises approximately 10% of lung cancer cases, more attention should be given to the therapeutic strategy for SCLC. Most SCLC cases respond to cytotoxic drugs, cisplatin, and etoposide. The objective response rate to the standard regimen is reported to be approximately 70% that is sufficient as standard therapy. However, almost all tumors recur and become refractory to chemotherapy which is the most important problem of this deadly disease.Recently, for the first time in several decades, ICIs have changed the standard therapy for SCLC. It must be emphasized that although ICIs paved the new way for SCLC therapy, more precise and effective therapy for SCLC is desired. Unfortunately, precise molecular mechanisms of SCLC are yet to be understood. Recent elaborate studies on the cell biology of SCLC uncovered several important aspects of molecular mechanisms. Gene profiling of cancer cells can be done using modern technology like next-generation sequencing (NGS). In this minireview, we describe the advances of modern technology in SCLC research and consider future therapeutic strategies based on the molecular mechanisms of SCLC.