【作者】 陈浩；

【导师】 崔治中；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2012， 博士

【摘要】 根据致瘤机制的不同，禽白血病/肉瘤病毒群各成员病毒被划分慢性转化型病毒和急性转化型病毒两类。慢性转化型病毒通过插入启动机制间接激活细胞原癌基因（c-onc）而引发肿瘤，潜伏期较长，一般超过90天；而急性转化型病毒则是通过直接启动自身基因组中携带的病毒肿瘤基因（v-onc）而导致急性肿瘤的形成，潜伏期一般仅在数周之内（Fadly and Nair，2008）。禽ALV-J的原型毒株HPRS-103是一种复制完整型病毒，其基因组中不含有任何病毒致瘤基因，在机体内不能快速诱发肿瘤的形成，在体外也不能转化鸡骨髓细胞（Payneet al.,1992）。而在1993年，Payne等从该株病毒感染的表现有髓细胞瘤的实验室病鸡中分离到了数株携带有肿瘤基因v-myc的急性转化型病毒，这些病毒大多可以转化体外的骨髓细胞，在鸡体上可导致多种急性肿瘤的发生，如髓细胞瘤、肾胚细胞瘤及肾腺瘤等，而其中的966株则得到了较为详尽的研究（Payne et al.,1993）。2000年，Venugopal等从自然感染J亚群病毒的患有成红细胞性白血病的鸡群中分离到若干株含v-myc的急性病毒，它们不仅可以诱发急性成红细胞增多病，而且还能导致髓细胞瘤和其他肿瘤的形成（Venugopal et al.,2000）。Fujinami肉瘤病毒（FSV）于1914年从日本分离出来（Fujinami and Inamoto,1914），起初曾认为它和带有v-src肿瘤基因的劳斯肉瘤病毒（Rous Sarcoma Virus，RSV）是同一病毒的不同毒株，后来经序列分析发现它是完全不同的另一种带有v-fps肿瘤基因的急性缺陷型病毒（Shibuya and Hanafusa,1982）。此外，PRCII是一株从自然发病鸡中分离的在5′-fps端缺失1020个核苷酸的此类型毒株，它转化细胞能力及致肿瘤能力稍弱（Carlberg et al.,1984; Huang et al.,1984）。近年来，我们发现在国内爆发该病的鸡群中出现了急性肉瘤病例，病例组织切片法检测表明其为典型纤维肉瘤，通过病毒分离试验确定病料中含有ALV-J（刘绍琼等，2011）。肉瘤组织研磨滤过液及其接种过的第一代DF细胞上清，在接种1日龄SPF鸡和817肉杂鸡后，可以在9天内复制出相同的纤维肉瘤（李传龙等，2012）。因此，我们怀疑其中除了含有完整复制型的ALV-J外，很可能还存在一种可以引发快速致瘤的携有某种病毒肿瘤基因（v-onc）的缺陷型病毒。本研究课题初步探究了缺陷型病毒在体外CEF细胞培养中的生物学活性；提取病料中的病毒RNA和前病毒DNA，使用PCR法筛选到相关v-onc；然后制备针对该基因所编码蛋白的特异性抗血清，建立免疫荧光方法、免疫组化法分别对感染细胞中的病毒抗原、肉瘤组织切片进行检测；构建含有该肿瘤基因的缺陷型病毒的全基因组质粒，拯救分子克隆化病毒，回归动物复制病征，从而在分子和蛋白质两个水平上佐证缺陷型病毒是造成急性纤维肉瘤的主因。为了观察肉瘤病料中缺陷型病毒在体外细胞培养中的增殖能力，分别将肉瘤组织滤过液及其感染后的细胞第1代毒、第2代毒接种相同的三组试验鸡只，结果发现第1代细胞毒同肉瘤研磨液具有相似的致瘤能力，但第3代细胞毒的致瘤速度及诱发鸡只出现肿瘤的比例则明显下降，这表明肉瘤中存在的缺陷型病毒粒子在体外CEF细胞培养过程中，产生的子代病毒粒子会逐渐减少，结合其不能引起CEF出现细胞病变效应的现象，我们推测该类缺陷型病毒很可能不适应在CEF上生长。使用PCR方法，我们扩增到了复制完整型ALV-J病毒粒子的完整基因组（命名为SD1005），同国内外其他12个J亚群毒株比对发现，它与引发慢性髓细胞瘤和血管瘤的毒株JS09GY6（Wu et al.,2010）具有相似的基因组结构，且整体核酸同源性高达97%，与其它毒株相比，亦没有显著的基因缺失或突变。而且，其基因组结构中并不含有任何病毒肿瘤基因，与J亚群病毒原型株HPRS-103同属于完整复制型病毒，再结合禽白血病/肉瘤病毒致瘤机制（Fadly and Nair，2008），可以排除SD1005是导致这种急性肿瘤形成的原因。随后，使用多对扩增不同肿瘤基因嵌合体分子的引物，我们确定了数个含有v-fps肿瘤基因的缺陷型基因组（Fu-J1~5），其结构特征为：5′-R-U5-△gag-fps-△env-U3-R-3′或5′-R-U5-△gag-fps-△pol-△env-U3-R-3′。在env基因水平上，Fu-Js同SD1005的同源性超过99%，这说明缺陷型Fu-Js很可能起源于作为辅助病毒的SD1005。值得注意的是，尽管Fu-Js中的v-fps基因同Fujinami和PRCII株的v-fps基因具有超过98%的核酸同源性，但不同的是，FSV和PRCII二者在该基因的5′和3′完全一致，而我们所扩增到的所有序列在5′端则相对缺少24碱基（但与gag基因的结合位点稳定），在3′则有200~500nt的缺失，长度较多样化，且插入位点也不稳定。使用DNAstar软件分析并筛选两段抗原性较好的截短fps基因，连接到pET32a（+）载体中，经E.coli表达系统获得人工Fps蛋白，免疫后得到鼠抗Fps多克隆血清，建立间接免疫荧光方法并检测肉瘤组织悬液感染的CEF细胞，结果呈现阳性，表明急性肉瘤中确实存在一种可以编码Fps相关蛋白的缺陷病毒。同时，建立免疫组织化学方法检测纤维肉瘤组织切片，发现在细胞质中存在Fps肿瘤蛋白。选取Fu-J1和Fu-J3两个基因组，通过重叠延伸PCR技术（SOE-PCR）和限制性内切酶酶切技术分别获得质粒pTZ57-Fu-J1和pTZ57-Fu-J3。参考Fujinami基因组，合成一段fps序列，将缺少219nt的3′-fps段添补到质粒pTZ57-Fu-J1中，构建了另外两个质粒pTZ57-Fu-J1E和pTZ57-Fu-J1M。将这4种质粒转染到CEF细胞中，使用制备的抗Fps血清，可以检测到阳性细胞的存在，但是以其上清再次接种新的CEF，却不能检测到荧光的存在，这表明尽管质粒的构建是成功的，能够产生瞬时表达，但却不能拯救到有传染性的分子克隆化病毒。正如同体外传代试验所证明的那样，该病毒很可能不适合在体外CEF中培养。而使用质粒DNA直接接种SPF鸡的试验中，有1只鸡在接种后19天后在接种部位出现了肉瘤肿块，并证实是纤维肉瘤。我们推测，反转录病毒ALV-J在与鸡体原癌基因的重组过程中可能会产生多个在基因水平上有差异个体，它们形成一个准种群体（quasispecies），而其中仅有一个或是数个具有生物学活性，而其他的则是无效基因体。我们的试验结果表明，含有v-fps的相关缺陷型J亚群白血病病毒是诱导急性纤维肉瘤的直接原因，这也暗示ALV-J诱导肿瘤形成的机制中，除了是激发已经证明的myc基因外（Chesters et al.,2000），很可能还包括fps基因。更多还原

【Abstract】 Avian Leukosis/Sarcoma Viruses (AL/SVs) had been divided into non-acute transformingvirus and acute transforming virus in accordance with different oncogenesis mechanisms.Non-acute transforming virus caused tumors after a long latent period of over90days byinsertional activation of cellular proto-oncogene, while acute transforming virus could inducea fast tumorigenesis within several weeks through direct transcription of viral oncogene(Fadly and Nair,2008).The HPRS-103strain, prototype of ALV-J, has a genome without any viral oncogene,neither cause tumors rapidly in vivo and nor transform myeloid cell in vitro (Payne et al.,1992). However, acutely transforming viruses with v-myc oncogene had been isolated fromcases of myelocytomatosis induced experimentally by HPRS-103(Payne et al.,1993), mostof them can transform cultured chicken bone marrow cells in vitro and induce a variety ofacute tumors including myelocytomatosis, nephroblastoma, renal adenomas/adenocarcinomasin chickens. Lately, acutely transforming ALV-J strains with v-myc have also been isolatedfrom naturally infected parent flocks (Venugopal et al.,2000), although they mainly inducederythroblastosis as well as myelocytomatosis.Fujinami Sarcoma Virus (FSV) was discovered in Japan (Fujinami and Inamoto,1914).Previous studies have shown that it has a genome with v-fps oncogene which is different fromv-src in Rous Sarcoma Virus (RSV), although both of them can cause acute fibrosarcomas inchickens (Shibuya and Hanafusa,1982). PRCII is a naturally obtained5′-fps deletion of1020nucleotides mutant, showed low efficiency of transforming activity on cells and decreasedcarcinogenicity in chickens (Carlberg et al.,1984; Huang et al.,1984).Over the past few years, we have observed the occurrence of lesions indicative offibrosarcoma in tissues from parent layers and hybrid chickens (cross-breeding between commercial female layers and white meat-type male) in China. Researchers in our lab havemade a definite diagnosis of acute fibrosarcoma in these chickens, which is characterized byrapid growth and infiltration into surrounding tissues and metastasis, and demonstrated that itis associated with the infection of ALV-J (Liu et al.,2011; Li et al.,2012). The same acutefibrosarcoma occurred at the9thday after the filtered supernatant of sarcomas and the1stpassage DF1infected by sarcoma supernatant. So we suspect that there exists some certaindefective virus with viral oncogene in the fibrosarcoma.This study preliminarily explored the biological activity of defective virus in vitro CEFcell culture; extracted the viral RNA and proviral DNA, then usd the PCR method to screenthe relevant v-oncogene succesfully; prepared artificial protein encoded by v-fps and specificserum against Fps protein, established the indirect immunofluorescence assay for detection ofviral antigens in infected cells and demonstrated the existence of oncoprotein; constructed4recombinant plasmid carrying v-fps, then injected them into SPF chicken, the similar tumorwas observed. We provided the evidence to prove that the defective viruses Fu-Js were thereal reason to cause acute fibrosarcomas from two perspects of gene and protein.In order to observe the productivity of defective virus cultured in vitro CEF, tumorsupernatant and the1st,3rdpassage of CEF cell infected by it were inoculated respectively into10SPF chickens. The results indicated that the1stpassage of CEF had the same strongtumorigenicity as the tumor supernatant, while the3rdpassage of CEF had a remarkabledecreased tumorigenic ability. It suggested that progeny virus particles may gradually reducedin the process of vitro culture. Combined with the absence of CPE, we inferred that this typeof defective virus is likely not suited to grow in CEFs.PCR amplified the whole genome sequence of the replication-competent ALV-J (SD1005),alignment of the complete sequence demonstrated that it has no remarkable mutation in thegenome and appears not to be the real pathogen inducing the acute tumorigensis. Additionally,we obtained several genome sequences associated with ALV-J (Fu-J1~5), which is defective,with parts of gag, pol and env genes replaced by a sequence of fps gene. The sequences ofFu-Js revealed that their genomes are closely related to that of SD1005(Wu et al.,2010), and probably derived from it in accordance with the high homology (＞99%) in env gene betweenthem. Notably, compared with homologous region of Fujinami and PRCII, the obtained v-fpsgenes in our study have two deletion mutations at both ends, with the major diversity at the3′-fps end especially.Anti-Fps polyclonal antiserum was prepared by standard methods by references. Aprokaryotic expression vector containing v-fps oncogene was constructed and inverted intoEscherichia coli (E. coli) expression system. Balb/c mouse were immunized with purifiedproteins expressed from Escherichia coli (E. coli) and antiserum was monitored by ELISAmethod. These antisera was shown to be special, sensitive and useful in detection tests (datasin press). CEF cells inoculated with tumor supernatant were detected by the indirectfluorescence assay (IFA) with anti-Fps polyclonal antisera. The result was positive, implyingthat natural Fps oncoprotein indeed existed in the infected cells.Plasmid pTZ57-Fu-J1and pTZ57-Fu-J3were obtaind successfully by SOE-PCR andrestriction endonuclease technology. Reference to Fujinami genome, pTZ57-Fu-J1E andpTZ57-Fu-J1M were acquired by the restruction of pTZ57-Fu-J1by the addition of219nt at3′-fps end. These four plasmids were transfected into CEFs, and detected by IFA with theanti-Fps serum as the first antibody. The positive cell can be observed, suggesting thattransient transfection was successful. However, the infectious molecular clone of virus can notbe saved. In the experiment of direct inoculation into SPF chickens of plasmid DNA, sarcomacan be detected in one chicken at the19thdays after inoculation, despite of the low incidenceratio, but it had already demonstrated that the constructed plasmid has the ability to inducetumors to some extent.So we infer that it seems to be unstable for junction site in the process of recombinationbetween c-fps oncogene and retrovirus ALV, a majority of variable individuals at gene level(quasispecies) will generate, one strain of which has biological activity at least. Obviously, theinduction of acute fibrosarcomas in our experiment occurs through mechanisms involving thedirect activation of viral oncogene v-fps in Fu-Js rather than insertional activation of cellularoncogene c-fps, revealing that the tumorigenesis induced by ALV-J is probable to involve fps oncogene besides for the myc oncogene (Chesters et al.,2000). This is the first report on acutefibrosarcomas caused by ALV-J containing v-fps oncogene since the identification of thisvirus in1988.更多还原