CN109022470A - J亚群禽白血病病毒gp85基因重组原核表达蛋白及其纯化方法和应用 - Google Patents
J亚群禽白血病病毒gp85基因重组原核表达蛋白及其纯化方法和应用 Download PDFInfo
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Abstract
本发明公开了一种J亚群禽白血病病毒gp85基因重组原核表达蛋白及其纯化方法和应用，包括下列步骤：（1）设计并合成扩增gp85基因的特异性引物（2）PCR扩增获得gp85基因片段并纯化；（3）将gp85基因克隆至pET‑32a原核表达载体，筛选阳性克隆后测序鉴定；（4）重组表达载体pET‑32a‑gp85的表达；（5）尿素法纯化表达的gp85蛋白；（6）重组gp85蛋白的复性。利用本发明的方法获得了高纯度的可溶性gp85蛋白。表达的蛋白可作为包被抗原建立抗体检测方法，还可用于免疫动物制备抗血清，为免疫荧光、免疫组化、胶体金检测卡制备等提供原材料。
Description
技术领域
本发明涉及生物技术领域，特别是涉及一种J亚群禽白血病病毒gp85基因重组原核表达蛋白及其纯化方法和应用。
背景技术
1991年，自英国科学家Payne首次报道J亚群禽白血病病毒（avian leukosisvirus subgroup J, ALV-J）后，在世界各大洲的肉种鸡群中全面爆发该病，尤其是1997和1998年使世界养禽业遭受了毁灭性打击。由于ALV-J在养鸡业的广泛流行、我国养殖模式的多样化、规模参差不齐等特点，进行ALV-J的净化还是一项巨大的挑战。目前无有效的疫苗和药物进行该病的防治，临床上主要靠ELISA抗原/抗体试剂盒检测后剔除阳性鸡来控制感染。现在开展血清学监测通常使用美国等公司的检测试剂盒，检测成本高、推广范围窄。根据国外毒株研制的检测方法和试剂盒不能准确检测到国内鸡群的感染状况，因此建立快速检测ALV-J的方法对国内ALV-J的净化尤为重要。ALV-J的env基因编码病毒囊膜糖基化蛋白，包括膜表面糖蛋白亚单位gp85和跨膜糖蛋白亚单位gp37。gp85蛋白是病毒的主要抗原，病毒中和反应的作用位点，含有病毒-受体决定簇，主要起病毒粘附易感动物细胞的作用，同时也能诱导有免疫力的鸡群产生特异性抗体。如何将ALV-J的重组gp85蛋白进行原核表达并纯化得到纯度高可溶性好的gp85蛋白，是本发明要解决的技术问题。
发明内容
本发明就是针对上述存在的缺陷而提供一种J亚群禽白血病病毒gp85基因重组原核表达蛋白及其纯化方法。本发明将ALV-J分离毒株的重组gp85蛋白进行原核表达并利用尿素法纯化得到包涵体蛋白，通过透析复性得到了纯度高可溶性好的gp85蛋白。表达的蛋白可作为包被抗原建立抗体检测方法，还可用于免疫动物制备抗血清，为免疫荧光、免疫组化、胶体金检测卡制备等提供原材料。同时，利用单克隆抗体技术制备抗蛋白的单克隆抗体，为今后对该病毒的临床诊断和抗原检测试剂盒的开发提供物质基础。
本发明的J亚群禽白血病病毒gp85基因重组原核表达蛋白及其纯化方法技术方案为，
一种J亚群禽白血病病毒gp85基因重组原核表达蛋白的纯化方法，包括以下步骤：
（1）设计扩增gp85基因的特异性引物：
ALV-J-F：5’- CG GGATCC GGAGTTCATCTGTTGCAACAACCAG-3’（加粗代表保护碱基，斜体为BamHⅠ酶切位点）
ALV-J-R：5’- CCC AAGCTT TTA GCGCCTGCTACGGCGGTGAC-3’（加粗代表保护碱基，斜体为HindⅢ酶切位点，下划线为终止密码子）；
（2）PCR扩增获得gp85基因片段并纯化；
（3）将gp85基因克隆至pET-32a原核表达载体，筛选阳性克隆后测序鉴定；
（4）重组表达载体pET-32a-gp85的表达；
（5）尿素法纯化表达的gp85蛋白；
（6）重组gp85蛋白的复性。
步骤（2）中，应用2×Pfu PCR Mix进行PCR扩增，反应程序为：95 ℃预变性5 min，95 ℃变性30 s，55 ℃退火30 s，72 ℃延伸1 min，共30个循环，最后72 ℃延伸7 min。
步骤（3）中，gp85基因片段及pET-32a空载体分别用BamHⅠ、HindⅢ快速内切酶进行双酶切，37 ℃温浴15 min，纯化双酶切后的目的基因和载体片段，配置10 μL连接体系，4℃反应过夜；连接产物转化E.coli Rosetta (DE3)感受态，涂氨苄青霉素LB平板后，37 ℃温箱孵育过夜；挑取LB平板上单个的白色菌落，接种于5 mL含Amp的LB液体培养基中，37 ℃培养10 h，提取质粒。
步骤（4）中，表达条件：30℃，摇床转速为160 rpm，终浓度为0.1 mM的IPTG，诱导5h。
步骤（5）具体为，用溶液1重悬菌体，超声波冰浴裂解，再通过溶液2、溶液3、溶液4洗涤，溶液5溶解包涵体，装入处理过的透析袋中，以TE buffer溶解的4M尿素溶液为初始透析液，通过逐渐稀释透析液的方法使透析袋中的尿素浓度依次降低，至袋中尿素浓度为零时收获复性蛋白；
溶液1：50 mmol/L Tris-Hcl pH=8.0，1 mmol/L EDTA pH=8.0，100 mmol/L NaCl；
溶液2：500 mmol/L Tris-Cl pH=8.0，100 mmol/L NaCl，1 mmol/L EDTA，0.5 %TritonX-100；
溶液3：溶液2含2 M尿素；
溶液4：溶液2含4 M尿素；
溶液5：溶液2含8 M尿素。
所要表达的gp85基因编码的核苷酸序列如SEQ ID NO：1所示：
GVHLLQQPGNVWVTWANKTGQTDFCLSLQSATSPFRTCLIGIPQYPLSTFEGYVANITACKNDADLANQTACLIQTLNTTLPWDPQELDILGSQMIKNGTTRTCVAFGSVCYKENSSTVCHIFDGNFNGTGGPEAELRDFITKRKGNDHLIRPYVNQSWAMVSPINTESFSISSRYCGFTSNETRYYPGNQSVFCSSKGGEWSPAYRNGTQCSNNTTGCGSNCTAEWNYYAYGFTFGNKPEVLWNNGTAKALPPGIFLICGDRAWQGIPSNALGGPCYLGQLTMLSPNFTTWMTYGPNITGHRRSRR。
所述的纯化方法得到的J亚群禽白血病病毒gp85基因重组原核表达蛋白。
所述的J亚群禽白血病病毒gp85基因重组原核表达蛋白在检测J亚群禽白血病病毒上的应用。
所述的J亚群禽白血病病毒gp85基因重组原核表达蛋白在制备J亚群禽白血病病毒抗体上的应用。
本发明的有益效果为：
（1）利用大肠杆菌为表达宿主菌，其结构简单，生长速度快，易于培养和发酵，有效降低了生产成本。表达量高且表达条件易于标准化适用于规模化生产。
（2）纯化试剂主要为不同浓度的尿素溶液，价格低廉，操作简单，纯化后的蛋白纯度高。
（3）gp85蛋白是病毒囊膜表面的主要抗原，表达的蛋白可作为包被抗原建立抗体检测方法，还可用于免疫动物制备抗血清，为免疫荧光、免疫组化等提供原材料。该发明提供的包涵体蛋白纯化及变复性方法为相关单位进行gp85蛋白的原核表达提供了方法。
（4）实验表明，本发明的方法可用于J亚群禽白血病病毒不同毒株gp85蛋白的表达和纯化。
附图说明：
图1所示为获得的gp85基因及重组质粒pET-32a-gp85双酶切鉴定的电泳图：图中，M为DL5000bp Marker，1为gp85基因PCR扩增产物，2为重组质粒pET-32a-gp85双酶切鉴定获得的两条特异性条带；
图2所示为重组gp85蛋白在不同温度、时间、IPTG浓度条件下表达的SDS-PAGE电泳图：图中，M为蛋白质Marker，1，2，3分别代表在25℃，30℃，37℃条件下的蛋白表达情况；4，5，6，7分别代表诱导时间为3h，4h，5h，6h条件下的蛋白表达情况；8，9，10，11分别代表诱导剂浓度为0.1 mmol/L，0.5 mmol/L，1.0 mmol/L，2.0 mmol/L条件下的蛋白表达情况；
图3所示为纯化复性后重组gp85蛋白的SDS-PAGE电泳图和Westernblot鉴定结果：图中，M为彩色预染蛋白质Marker，1为gp85蛋白，2为ALV-J感染鸡临床阳性血清为一抗进行Western blot鉴定gp85蛋白的反应原性。
具体实施方式：
为了更好地理解本发明，下面用具体实例来详细说明本发明的技术方案，但是本发明并不局限于此。
实施例１
J亚群禽白血病病毒gp85基因重组原核表达蛋白及其纯化方法
1、实验材料
1.1质粒载体和工程菌
原核表达载体pET-32a、E.coli Rosetta(DE3)表达菌
1.2试剂
2×Pfu PCR MasterMix、DNA Marke DL2000/DL5000、预染蛋白标准Maker 10-180 KD为北京康润诚业生物科技有限公司（GenStar）产品；BamHⅠ、HindⅢ快速内切酶为北京全式金生物技术有限公司产品（TransGen）；HRP标记的羊抗鸡二抗为SIGMA产品；普通质粒小提试剂盒、琼脂糖凝胶DNA回收试剂盒为OMEGA产品；氨苄青霉素、磷酸盐药品、碳酸盐药品、无水乙醇、SDS、甘油、尿素等试剂均为国产化学分析纯产品。
2、实验方法
2.1主要试剂配制
(1) LB培养基：称取胰蛋白胨10 g，酵母提取物5 g，NaCl 10 g溶于800 mL去离子水中，调节pH值为7.0左右后定容至1000 mL。制备固体培养基时需加入琼脂使其终浓度为1.5%，高压灭菌后倒板。
(2) 氨苄青霉素（Amp）溶液：浓度100 mg/mL，0.22 μm虑膜过滤除菌，-20 ℃保存备用。
(3) PBS缓冲液：NaCl 8.0 g，Na2HPO4·12H2O 3.58 g，KH2PO4 0.2 g，Kcl 0.2 g，待完全溶解后调液体PH为7.4，用蒸馏水定容至1000mL，高压灭菌后备用。
(4) 24 mg/mL IPTG：称取1.2 g IPTG至50 mL离心管中，加入40 mL去离子水，待完全溶解后定容至50 mL，0.22 μm虑膜过滤除菌，小份分装，-20 ℃保存。
(5) 溶液1：50 mmol/L Tris-Hcl PH 8.0，1 mmol/L EDTA PH 8.0，100 mmol/LNaCl
溶液2：500 mmol/L Tris-Cl（PH=8.0），100 mmol/L NaCl，1 mmol/L EDTA，0.5%TritonX-100
溶液3：溶液2含2 M尿素
溶液4：溶液2含4 M尿素
溶液5：溶液2含8 M尿素
(6) 初始透析液：1×TE溶液含4M尿素，加入甘油，终浓度为10%。
2.2 gp85基因的克隆
应用NCBI在线软件设计扩增ALV-J gp85基因的特异性引物，在上游引物中添加 BamHⅠ酶切位点，下游引物中添加HindⅢ酶切位点。引物序列为：
F: 5’- CG GGATCC GGAGTTCATCTGTTGCAACAACCAG-3’（加粗代表保护碱基，斜体为BamHⅠ酶切位点），R: 5’- CCC AAGCTT TTA GCGCCTGCTACGGCGGTGAC-3’（加粗代表保护碱基，斜体为HindⅢ酶切位点，下划线为终止密码子）。ALV-J BZ-0415毒株（申请人于2016年分离自滨州市某养殖场的海兰褐种公鸡）接种DF-1细胞后培养7天，收获细胞提取RNA后反转录为cDNA作为模板，应用2×Pfu PCR Mix进行PCR扩增，反应程序为：95 ℃预变性5 min，95 ℃变性30 s，55 ℃退火30 s，72 ℃延伸1 min，共30个循环，最后72 ℃延伸7 min。用OMEGA胶回收试剂盒纯化PCR扩增获得的目的基因片段，操作按试剂盒说明书进行。
2.3重组表达载体pET-32a-gp85的构建
胶回收后的gp85基因片段及pET-32a空载体分别用BamHⅠ、HindⅢ快速内切酶进行双酶切，37 ℃温浴15 min，胶回收试剂盒纯化双酶切后的目的基因和载体片段，配置10 μL连接体系，4 ℃反应过夜。连接产物转化E.coli Rosetta (DE3)感受态，涂氨苄青霉素LB平板后，37 ℃温箱孵育过夜。挑取LB平板上单个的白色菌落，接种于5 mL含Amp的LB液体培养基中，37 ℃培养10 h，按质粒小提试剂盒说明书操作提取质粒，用BamHⅠ、HindⅢ快速内切酶进行双酶切验证，阳性质粒送上海生工生物工程股份有限公司测序。
2.4 gp85基因的原核表达
(1) 测序后的阳性克隆用LB活化后冻存菌种。取10 μL菌液接种含Amp的5 mL LB培养基中，37 ℃摇床振荡培养过夜。次日，1:100重新接种于含Amp的5 mL LB培养基中，37 ℃振荡培养约2 h ( 菌液OD600达0.6-0.8 )，取1 mL未诱导的菌液作为阴性对照，其余菌液加入IPTG至终浓度1.0 mmol/L，振荡培养4 h。诱导后菌液3 mL，10000 r/min离心1 min，弃上清，1 mLPBS重悬沉淀，冰浴条件下超声波破碎菌体，200 W/次，超声3 s，间歇3 s，超声5min。对破碎后的菌液10000 r/min离心5 min，将上清转移至另一EP管中，沉淀用1 mL PBS重悬，各取上清和沉淀40 μL 加10 μL的5 × SDS上样缓冲液，混匀后沸水浴中煮5 min，10000 r/min离心5 min，取上清20 μL进行12 %SDS-PAGE电泳。未诱导菌液进行同样的处理，分析目的蛋白在菌体中的分布情况。
(2) 优化表达条件：在IPTG终浓度为1.0 mmol/L，诱导时间为4 h，诱导温度分别为25 ℃、30 ℃、37 ℃环境条件下表达，12 %SDS-PAGE电泳分析蛋白表达情况选择最适温度；在30 ℃，IPTG终浓度1.0 mmol/L，诱导时间分别为3 h、4 h、5 h和6 h时，12 % SDS-PAGE电泳分析蛋白表达情况选择最适诱导时间；在37 ℃，诱导时间为5 h，IPTG终浓度分别为0.1、0.5、1.0、1.5、2.0 mmol/L时，12 %SDS-PAGE电泳分析蛋白表达情况选择最适IPTG浓度；在30 ℃，IPTG终浓度为0.1 mmol/L，诱导5 h，摇床转速分别为100、120、150、160、180、200 rpm时，12 %SDS-PAGE电泳分析蛋白表达情况选择最适摇床转速。
2.5 gp85蛋白的纯化和复性
2.5.1 gp85蛋白的纯化
(1) 将pET-32a-gp85的表达宿主菌过夜培养物10 mL加入1L LB培养基中，置于37 ℃摇床中160 rpm活化约3 h（OD600约为0.8左右），加入终浓度为0.1 mM的IPTG诱导4 h。诱导后菌液10000 rpm离心5 min，弃上清。200mL PBS重悬洗涤，相同条件离心，弃上清。
(2) 用80 mL溶液1重悬菌体，400 w 超声4 s停4 s，超声20 min左右至菌液不再粘稠。12，000 rpm离心8 min，弃上清。
(3) 用150 mL溶液2重悬沉淀，静置10 min后，12000 rpm离心6 min后弃上清；用150 mL溶液3重悬沉淀，静置15 min后，4 ℃ 12000 rpm离心8 min后弃上清；用150 mL溶液4重悬沉淀，静置20 min后，4 ℃ 12000 rpm离心10 min后弃上清；按每100 mL原始菌液加4mL溶液5计算，共加入40 mL溶液5涡旋器震荡1 h助溶。
(4) 4 ℃ 12000 rpm离心10 min，取上清，即为溶解的包涵体蛋白。12 % SDS-PAGE检测纯化后gp85蛋白的纯度。
(5) 将纯化后的gp85蛋白经12 % SDS-PAGE电泳后转印至PVDF膜上，以ALV-J感染鸡临床阳性血清1:200倍稀释后为一抗，HRP标记的羊抗鸡IgG 1:2000倍稀释后为二抗，Western blot鉴定gp85蛋白的反应原性。
2.5.2包涵体的复性
12 % SDS-PAGE检测后将纯化蛋白的浓度用溶液5稀释为1 mg/mL装入处理好的透析袋中，放入1 L初始透析液中，采用透析复性的方法使透析袋中的尿素浓度依次降低：4 M-2M-1 M-0.5 M-0.25 M-TE，每次透析4 h。透析过程中注意是否有白色絮状物析出，如果有大量沉淀析出则包涵体复性未成功，若无则表示成功。用12 % SDS-PAGE检测复性后蛋白的纯度与浓度。复性好的蛋白经0.45 µm滤器过滤后分装1 mL/管保存于-80 ℃备用。
3、实验结果
3.1重组表达载体pET-32a-gp85的构建
构建的重组质粒命名为pET-32a-gp85，用EcoRI和NotI双酶切后，产生约为5.9 kb的载体骨架片段和921bp的gp85基因片段（图1）。重组质粒的测序结果表明，插入pET-32a质粒中的gp85基因片段大小为 921bp，包括完整基因ORF，含有六个组氨酸构成的His标签，终止密码子TTA，编码307个氨基酸。
3.2 gp85基因的原核表达
重组质粒小量表达后证明目的蛋白以包涵体的形式存在于超声破碎后的菌体沉淀中。通过实验优化了表达温度、表达时间、诱导剂浓度和摇床转速，结果表明，重组质粒的最佳表达条件为：30 ℃，诱导5 h，IPTG终浓度为0.1 mmol/L，摇床转速为160 rpm（图2）。
3.3 gp85蛋白的纯化和复性
gp85蛋白以包涵体的形式表达，通过冰浴超声破碎后弃掉上清，将含目的蛋白的沉淀依次用溶液2、3、4洗涤去除包涵体以外的杂蛋白，最后用含8M尿素的溶液5将包涵体蛋白溶解。采用透析复性的方法使透析袋中的尿素浓度依次降低，获得复性后的高纯度可溶性蛋白，Western blot结果表明纯化的gp85蛋白有良好的反应原性（图3）。
ALV-J是变异性很大的病毒，每个毒株的gp85蛋白都存在差异，本发明提供一种表达和纯化蛋白的实验方法，本领域技术人员能够根据需要来利用此方法表达gp85蛋白或他们想要表达的蛋白。
以下的3种ALV-J毒株同样能用该发明阐述的方法得到表达纯化后的gp85重组蛋白：
ALV-J HN0001毒株（GenBank:AY897219）2000年从白羽肉鸡分离得到
ALV-J NX0101毒株（GenBank: DQ115805）2001年从宁夏回族自治区某父母代种鸡场分离得到
ALV-J原型株HPRS-103（GenBank:Z36973）1989年英国的Payne从肉鸡中分离得到
实施例2
用纯化复性后的ALV-J gp85蛋白作为抗原免疫新西兰大白兔制备多克隆抗体。
多克隆抗体的制备及效价检测
1. 实验材料和方法
1.1 实验材料
ALV-J BZ-0415株病毒，白油Marcol52，DF-1细胞，FITC标记的羊抗兔二抗（SIGMA）。
1.2 免疫原制备
复性后的gp85蛋白与白油Marcol52按照1:3的比例乳化。
1.3 动物免疫
用3只1.5 Kg左右的新西兰大白兔来制备多抗，采用背部皮下多点注射，共免疫3次，200μg/只，每次间隔两周。3免后第7天将实验兔全身麻醉后用促凝的真空采血管通过颈动脉采血，37 ℃放置1 h后，4 ℃冰箱过夜分离血清。
1.4 间接免疫荧光反应（IFA）测定抗血清效价
将24孔细胞培养板中感染了BZ-0415株病毒的DF-1细胞依次用1×PBS洗1遍，丙酮：乙醇（3:2）固定液固定7 min，1×PBS洗3次。将制备的抗血清分别用1×PBS 按1:50、1:100、1:200、1:400、1:800、1:1000 六个稀释度加到固定好的DF-1细胞上，100μL /孔，37 ℃孵育45min，1×PBS洗3次。再加1:100稀释的FITC标记的羊抗兔IgG，100μL /孔，37 ℃孵育45 min，1×PBS洗3次。每孔加1 滴 50% 的甘油，在荧光显微镜下观察实验结果。
2. 实验结果
蛋白与白油混匀后呈乳白色乳剂，为油包水型。移液器吸取少量乳化后抗原滴于冷水中，除第1滴外，均不扩散。3只大白兔三免后共收集抗血清70 mL。
抗血清的IFA效价为1:400。
序列表
<110> 山东省滨州畜牧兽医研究院
<120> J亚群禽白血病病毒gp85基因重组原核表达蛋白及其纯化方法和应用
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序列表
<110>山东省滨州畜牧兽医研究院
<120> J亚群禽白血病病毒gp85基因重组原核表达蛋白及其纯化方法和应用
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Asn Lys Thr Gly Gln Thr Asp Phe Cys Leu Ser Leu Gln Ser Ala Thr
20 25 30
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Claims (9)
1.一种J亚群禽白血病病毒gp85基因重组原核表达蛋白的纯化方法，其特征在于，包括以下步骤：
（1）设计扩增gp85基因的特异性引物：
ALV-J-F：5’- CG GGATCC GGAGTTCATCTGTTGCAACAACCAG-3’
ALV-J-R：5’- CCC AAGCTT TTA GCGCCTGCTACGGCGGTGAC-3’；
（2）PCR扩增获得gp85基因片段并纯化；
（3）将gp85基因克隆至pET-32a原核表达载体，筛选阳性克隆后测序鉴定；
（4）重组表达载体pET-32a-gp85的表达；
（5）尿素法纯化表达的gp85蛋白；
（6）重组gp85蛋白的复性。
2. 根据权利要求1所述的J亚群禽白血病病毒gp85基因重组原核表达蛋白的纯化方法，其特征在于，步骤（2）中，应用2×Pfu PCR Mix进行PCR扩增，反应程序为：95 ℃预变性5min，95 ℃变性30 s，55 ℃退火30 s，72 ℃延伸1 min，共30个循环，最后72 ℃延伸7 min。
3. 根据权利要求1所述的J亚群禽白血病病毒gp85基因重组原核表达蛋白的纯化方法，其特征在于，步骤（3）中，gp85基因片段及pET-32a空载体分别用BamHⅠ、HindⅢ快速内切酶进行双酶切，37 ℃温浴15 min，纯化双酶切后的目的基因和载体片段，配置10 μL连接体系，4 ℃反应过夜；连接产物转化E.coli Rosetta (DE3)感受态，涂氨苄青霉素LB平板后，37 ℃温箱孵育过夜；挑取LB平板上单个的白色菌落，接种于5 mL含Amp的LB液体培养基中，37 ℃培养10 h，提取质粒。
4. 根据权利要求1所述的J亚群禽白血病病毒gp85基因重组原核表达蛋白的纯化方法，其特征在于，步骤（4）中，表达条件为：30℃，摇床转速为160 rpm，终浓度为0.1 mM的IPTG，诱导5h。
5. 根据权利要求1所述的J亚群禽白血病病毒gp85基因重组原核表达蛋白的纯化方法，其特征在于，步骤（5）具体为，用溶液1重悬菌体，超声波冰浴裂解，再通过溶液2、溶液3、溶液4洗涤，溶液5溶解包涵体，装入处理过的透析袋中，以TE buffer溶解的4M尿素溶液为初始透析液，通过逐渐稀释透析液的方法使透析袋中的尿素浓度依次降低，至袋中尿素浓度为零时收获复性蛋白；
溶液1：50 mmol/L Tris-Hcl，pH=8.0，1 mmol/L EDTA，pH 8.0，100 mmol/L NaCl；
溶液2：500 mmol/L Tris-Cl，pH=8.0，100 mmol/L NaCl，1 mmol/L EDTA，0.5 %TritonX-100；
溶液3：溶液2含2 M尿素；
溶液4：溶液2含4 M尿素；
溶液5：溶液2含8 M尿素。
6. 根据权利要求1所述的J亚群禽白血病病毒gp85基因重组原核表达蛋白的纯化方法，其特征在于，所要表达的gp85基因编码的核苷酸序列如SEQ ID NO：1所示：
GVHLLQQPGNVWVTWANKTGQTDFCLSLQSATSPFRTCLIGIPQYPLSTFEGYVANITACKNDADLANQTACLIQTLNTTLPWDPQELDILGSQMIKNGTTRTCVAFGSVCYKENSSTVCHIFDGNFNGTGGPEAELRDFITKRKGNDHLIRPYVNQSWAMVSPINTESFSISSRYCGFTSNETRYYPGNQSVFCSSKGGEWSPAYRNGTQCSNNTTGCGSNCTAEWNYYAYGFTFGNKPEVLWNNGTAKALPPGIFLICGDRAWQGIPSNALGGPCYLGQLTMLSPNFTTWMTYGPNITGHRRSRR。
7.如权利要求1-6任一所述的纯化方法得到的J亚群禽白血病病毒gp85基因重组原核表达蛋白。
8.如权利要求7所述的J亚群禽白血病病毒gp85基因重组原核表达蛋白在检测J亚群禽白血病病毒上的应用。
9.如权利要求7所述的J亚群禽白血病病毒gp85基因重组原核表达蛋白在制备J亚群禽白血病病毒抗体上的应用。
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