摘要
禽白血病(Avian Leukosis，AL)是由禽白血病／肉瘤病毒群病毒(Avian Leukosis／sarcoma virus，AL／SV)引起的禽类多种肿瘤性疾病的总称。本病在世界各地均有发生，感染率高，发病率低，是危害养禽业的主要疾病之一。流行病学调查研究表明，本病在我国也广泛的发生和流行。由于目前尚没有有效的疫苗和药物用于防治本病，世界各国控制禽白血病的主要措施是通过病原检测，淘汰阳性鸡，从而净化种群达到消灭本病的目的，因此建立一种快速准确的诊断方法就显得尤为重要。
迄今为止，国内外相继建立了多种禽白血病诊断方法，但其试剂制备利诊断过程均有不足之处。ALV衣壳蛋白p27是一种高度保守的非糖基化蛋白，在外源性ALV(A、B、C、D、J)各亚群间同源性高达90％，是ALV主要的群特异性抗原，且在病毒蛋白中含量最高，是禽白血病抗原检测的理想靶蛋白。本研究的目的是利用分子生物学技术克隆禽白血病衣壳蛋白p27基因，对p27基因进行原核和真核表达，为进一步研制快速准确、灵敏特异的抗原和抗体检测方法奠定基础。
从人工感染禽白血病劳斯肉瘤病毒(SR-RSV-E)的SPF鸡胚成纤维细胞(CEF)培养物中提取RNA，利用p27基因的特异性引物，通过RT-PCR方法扩增出720 bp的gag p27基因片段：将该片断克隆至pMD18-T载体，酶切鉴定后进行序列测定和分析，表明该片段的核苷酸序列与国外RSV分离株(J02342)的同源性达97.3％：将p27基因亚克隆至原核表达载体pGEX-6P-1，转化受体菌BL-21，经IPTG诱导表达，获得大小为56kD的可溶性融合蛋白，薄层扫描分析重组蛋白占菌体总蛋白的23％；纯化获得的重组p27蛋白具有良好的抗原反应活性，可代替全病毒用于间接ELISA诊断方法的建立。相比于通过提纯病毒，去污剂裂解，电泳纯化p27蛋白的传统方法，利用原核表达系统制备重组p27蛋白成本低，纯度高，周期短。对原核表达的重组p27蛋白用亲和层析法纯化后，免疫实验兔制备了抗血清。进而建立了琼脂扩散诊断方法，该方法简便、特异，可以满足现地使用的需要。
在原核表达的基础上，利用BAC-TO-BAC~(TM)杆状病毒表达系统对p27蛋白进行真核表达，所表达的产物能与全病毒制备的抗血清发生特异性反应。相比于原核表达系统，杆状病毒表达系统表达的蛋白具有与天然蛋白更相似的结构和功能，为下一步利用重组蛋白直接制备单克隆抗体创造了条件。单克隆抗体的研制为结合上述制备的p27蛋白多克隆抗体，建立一种简便快捷、适用于大面积推广的禽白血病／肉瘤病毒群抗原检测的双抗夹心ELISA创造了条件，必将为我国禽白血病的种群净化提供技术支持。
此外，表达ALV p27基因重组杆状病毒的获得，进一步结合表达ALV囊膜基因重组杆状病毒的构建，共感染昆虫细胞有望形成ALV病毒样颗粒，为疫苗的研制开创新的思路。
Avian Leukosis is a malignant neoplasitc disease caused by avian leucosis/sarcoma virus. Infection with ALV is widespread in poultry flocks in the world with high morbidity. Although sporadic cases of neoplasitc disease occur in most flocks and the incidence of death is usually low, heavy losses from AL are uncommon. Based on Epidemiological data, AL is highly prevalent and occurs ubiquitously in China. Coupled with the fact that vaccines or medicines against avian leucosis virus have not been successful up to now, AL was controlled primarily by the successful application of ALV eradication program to chickens. The eradication of exogenous avian leukosis virus (ALV) from infected lines of chickens depends on the exclusion of congenitally infected chicks from groups of hatched chicks and the prevention of reinfection from extraneous sources. Therefore obviously desirable, the development of a rapid, and exact diagnostic technique for detecting exogenous virus has been necessary precondition to control ALV.
At present, serologic diagnosis of ALV infection has been developed by variety methods. These test, however, have some disadvantages in practical application. The capsid protein p27, primary group specific antigen of ALV, is main component in virion. Amino acid sequence of p27 protein shares an about 90% identity between exogenous ALV subgroup A, B, C, D, J. Therefore, p27 is the ideal target protein for detecting ALV antigen. The purpose in this study was to clone and express ALV p27 gene in prokaryotic and eukaryotic cell, Further, to develop a rapid, exact, sensitive and specific diagnostic technique for detection of ALV antigen and antibody using recombinant p27 as antigen.
RNA was extracted from CEF cell artificially infected with avian leucosis SR-RSV-E and the gag-p27 gene was amplified by RT-PCR with p27 specific primers. Then the gene was cloned into plasmid pMD18-T and sequenced. Nucleotide sequence analysis indicated that the identity between cloned p27 gene and that published in GenBank (J02342) was over 97.3%. The whole coding region of p27 gene was subcloned into prokaryotic expression vector pGEX-6p-l. Then the recombinant plasmid carrying p27 gene was transformed into BL-21 E.coli. Through induced with IPTG, a soluble recombinant protein with molecular weight of 56kD expressed in E.coli was identified by SDS-PAGE. The content of recombinant p27 protein was 23% of the total cellular proteins at optimal conditions. The recombinant p27 was purified with Glutathion Spharose 4B affinity chromatography. Western-blot verified the antigen reactivity of recombinant p27 protein and an indirect Enzyme-Linked Immunosorbent Assay (E1ISA) will be developed using recombinant p27 a
s antigen for the detection of ALV infection in place of the whole ALV Compared with the former method for purification of p27, that is through extraction, disruption and Polyacrylamide gel electrophoresis (PAGE) of whole ALV, Antiserum was produced from rabbit immunized with purified recombinant p27 protein, then, An agar gel immune precipitation test (AG1P) using p27 antiserum as antibodies was developed to detect ALV antigen in the feather pulp of infected chickens, which was a simple and specific method suited for practical application.
Further, in this study, p27 gene was expressed in Sf9 insect cells by BAC-TO-BAC?expression system. And the antigenic reactivity of recombinant p27 protein was identified by Western blot with antiserum against whole ALV. The recombinant p27 protein obtained from insect cells was more similar to nature p27 protein in structure and function than that from prokaryotic cells. With purified recombinant p27 protein as antigen, monoclonal antibodies against p27 will be produced afterward. Based on the monoclonal antibodies against p27 and available polyclonal antibodies against p27 above-mentioned, a DAS-EL1SA will be developed to detect group specific antigen p27 from chicken infected with ALV. This DAS-ELISA would be more specific, sensitive and applicable for the detection for ALV antigens in a large
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