CN110372794A - 中华鳖类固醇生成因子-1多克隆抗体的制备方法及应用 - Google Patents
中华鳖类固醇生成因子-1多克隆抗体的制备方法及应用 Download PDFInfo
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- shelled turtle
- trionyx sinensis
- turtle trionyx
- polyclonal antibody
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Abstract
本发明实施例公开了一种中华鳖中华鳖类固醇生成因子‑1多克隆抗体的制备方法及应用，所述中华鳖中华鳖类固醇生成因子‑1多克隆抗体的制备方法包括(1)中华鳖Sf‑1基因的克隆；(2)中华鳖Sf‑1基因重组表达载体的构建；(3)中华鳖Sf‑1重组蛋白的诱导表达和纯化及Western Blot进行特异性分析；(4)多克隆抗体的制备。本发明采用分子生物学和基因工程的方法，构建了中华鳖Sf‑1基因的重组表达载体，诱导表达，亲和层析获得纯化的重组表达蛋白。本发明的方法制备的中华鳖Sf‑1多克隆抗体可用于免疫组化、免疫印迹实验要求，建立体外免疫分析、研究中华鳖Sf‑1的功能以及中华鳖免疫荧光染色激光共聚焦显微观察，还可用于龟鳖类、爬行类、鸡的类固醇生成因子‑1的免疫检测。
Description
技术领域
本发明实施例涉及基因工程领域，具体涉及一种中华鳖类固醇生成因子-1多克隆抗体的制备方法及应用。
背景技术
类固醇生成因子1(Sf-1)又被称为肾上腺4-结合蛋白基因(Ad4Bp)，属于核受体超家族中的一员，在类固醇合成、性腺发育、性别决定过程中具有重要作用。序列分析表明Sf-1具有保守的HoLI结构域，研究发现Sf-1在下丘脑、垂体、肾上腺、性腺、脾脏、肌肉等组织中均有表达。
哺乳动物研究中发现Sf-1主要参与类固醇激素合成，可以通过细胞色素P450类固醇羟化酶和3β-羟基类固醇脱氢酶作用调控类固醇生成。垂体组织中的Sf-1还可以通过激活促性腺激素释放激素受体(GnRH-R)、LHβ等基因的表达来影响性腺功能。在Sf-1缺陷型小鼠研究中发现Sf-1缺失会导致性腺发育不完全，出现雄性小鼠雌性化现象，表明Sf-1可能在精巢发育过程中发挥重要作用。
中华鳖(Pelodiscus sinensis)裙边胶原蛋白含量较高，营养丰富，是我国名贵淡水养殖品种之一，具有较高的营养价值。在人工养殖过程中发现雄鳖的生长速度明显快于雌鳖，相同培养条件下，雄性中华鳖均重约为雌性的1.4倍，培育全雄鳖是育种工作的重要目标。深入研究中华鳖性别决定与分化机制，有助于建立提高雄鳖孵化率或全雄鳖培育方法，对中华鳖养殖业开展具有重要意义。
从中华鳖中克隆获得Sf-1基因，分析其在精巢中的表达，同时制备多克隆抗体及免疫组化实验，对进一步研究Sf-1在精巢中的功能具有重要意义。
发明内容
为此，本发明实施例提供一种中华鳖类固醇生成因子-1多克隆抗体的制备方法及应用，以解决上述问题。
为了实现上述目的，本发明实施例提供如下技术方案：
根据本发明实施例的第一方面，提供一种中华鳖Sf-1多克隆抗体的制备方法，包括以下步骤：
步骤(1)中华鳖Sf-1基因的克隆：
按照Trizol试剂说明书提取中华鳖总RNA，用M-MLV反转录酶合成cDNA第一链，于-80℃保存；根据GenBank数据库中已报道的Sf-1基因序列，设计引物，进行PCR扩增，获得中华鳖Sf-1基因cDNA片段；根据Smart RACE试剂盒说明书，进行5′RACE-PCR和3′RACE-PCR扩增，获得中华鳖Sf-1基因cDNA片段的5′末端和3′末端，拼接获得其全长cDNA序列；中华鳖Sf-1基因的核苷酸和氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示；
步骤(2)中华鳖Sf-1基因重组表达载体的构建：
根据中华鳖Sf-1基因全长cDNA序列，设计一对特异性引物Sf-1-F1(5′-CGCGGATCCATGACGCCCGTCGACTACGAC-3′)和Sf-1-R1(5′-CCGCTCGAGCGCCAGTATCCGAGCCTTGA-3′)，分别在上、下游引物添加了BamHⅠ和XhoⅠ酶切位点；以中华鳖cDNA为模板，Sf-1-F1和Sf-1-R1为引物，进行PCR扩增；将PC R产物和pET-32a(+)表达载体进行双酶切，回收纯化，连接转化大肠杆菌DH5α，获得重组表达载体pET-Sf-1；
步骤(3)中华鳖Sf-1重组蛋白的诱导表达和纯化及Western Blot特异性分析：
将步骤(2)得到的重组表达载体pET-Sf-1转化大肠杆菌BL21(DE3)，在含有100mg/mL氨苄抗性的LB培养液中培养至OD值为0.6时，加入IPTG进行诱导，培养4h。离心收集菌体，PBS重悬，超声波破碎，然后加入5×上样缓冲液进行12％SDS-PAGE电泳检测目标蛋白的表达情况；
以1％的接种量将能表达重组蛋白的BL21添加到含有100mg/ml氨苄抗性的LB培养基中，大量培养，诱导表达；将诱导好的菌液离心，收集菌体用变性结合缓冲液溶解，超声波破碎，再离心收集上清，上样、结合、洗脱、透析，获得纯化的中华鳖Sf-1重组蛋白；SDS-PAGE电泳检测纯化后的中华-鳖Sf-1重组蛋白；
取30μg重组总蛋白进行SDS-PAGE电泳，然后将蛋白转至PVDF膜上。用带His标签的鼠抗作为一抗，用HRP标记的羊抗鼠IgG为二抗进行多抗的特异性检测；
步骤(4)多克隆抗体的制备：
将步骤(3)纯化的中华鳖pET-Sf-1重组蛋白作为抗原，对小鼠进行5次免疫，按体积1:1的比例加入弗氏完全佐剂，采用腹部皮下多点注射，免疫剂量为0.1mg；加强免疫选用弗氏不完全佐剂；末次免疫10天后，进行全血分离，免疫血清储存于-80℃备用；
步骤(5)免疫组化分析：
采用石蜡包埋法包埋中华鳖精巢组织，以5μm厚度进行切片；常规脱蜡后，用50μL的H2O2(3％)于37℃孵育10min，阻断内源过氧化物酶；将切片放入柠檬酸盐缓冲液中并加热至沸腾，20min后自然冷却；用5％牛血清蛋白进行封闭后加入100倍稀释的鼠抗Sf-1抗血清37℃温育2h，用PBS洗涤3次，加入HRP标记的山羊抗鼠IgG二抗，37℃孵育30min，DAB显色；苏木精复染、脱水透明及树胶封片。阴性对照以阴性血清代替一抗；
步骤(6)多克隆抗体的效价测定：
以纯化的重组蛋白为包被抗原，以第一次免疫前采集的鼠血清为阴性对照，将鼠抗Sf-1抗体进行1:100至1:2430000倍比稀释，以HRP标记的羊抗鼠IgG为二抗，以TMB为底物显色液，进行间接ELISA分析；以鼠抗Sf-1抗体OD 450nm值与阴性对照血清OD450nm值的比值≥2时为阳性作为判定标准，制备的鼠抗Sf-1抗体最大稀释度作为该抗体的有效效价。
本发明与现有技术相比具有如下有益效果：
1)本发明采用分子生物学和基因工程的方法，构建了中华鳖Sf-1基因的重组表达载体，诱导表达，亲和层析获得纯化的重组表达蛋白。
2)本发明的方法制备的中华鳖Sf-1多克隆抗体具有很强的特异性，可用于免疫组化、免疫印记等实验要求，建立体外免疫分析，为深入研究中华鳖Sf-1的功能提供一个重要工具。
3)本发明制备的中华鳖Sf-1多克隆抗体，可用于中华鳖免疫荧光染色激光共聚焦显微观察。
4)本发明制备的中华鳖Sf-1多克隆抗体，可能在其他龟鳖类Sf-1的免疫检测中发挥作用。
附图说明
为了更清楚地说明本发明的实施方式或现有技术中的技术方案，下面将对实施方式或现有技术描述中所需要使用的附图作简单地介绍。显而易见地，下面描述中的附图仅仅是示例性的，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图引伸获得其它的实施附图。
图1和图2为中华鳖Sf-1抗原片段的克隆结果及重组表达载体的酶切检测结果；
图3为中华鳖Sf-1基因小量表达和纯化重组蛋白SDS-PAGE电泳图；
图4为中华鳖Sf-1蛋白特异性检测图；
图5和6为中华鳖精巢组织的抗血清免疫组化分析；
图7为鼠抗Sf-1血清的效价测定结果示意图。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
本实施例提供一种中华鳖Sf-1多克隆抗体的制备方法，包括以下步骤：
步骤1中华鳖Sf-1基因的克隆：
按照Trizol试剂说明书提取中华鳖总RNA，利用M-MLV反转录酶合成cDNA，cDNA第一链的合成根据Smart RACE试剂盒说明书进行，于-80℃低温保存。根据GenBank数据库已报道的Sf-1基因序列进行同源比对，设计特异性引物Sf-1-F(5′-GAATGCGACTAGAAGCTGTGCG-3′)和引物Sf-1-R(5′-GCAGGCTGAAGAGGATCAGGAA-3′)。以中华鳖cDNA第一链为模板，进行PCR扩增，获得中华鳖Sf-1基因cDNA片段；特异性引物Sf-1-F与Sf-1-R的核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。
PCR反应程序：
根据所得中华鳖Sf-1基因cDNA片段设计特异性引物Sf-1-GSP5′(5′-TCGGACTTGATGGTGCGGTTGGGA-3′)和Sf-1-NP5′(5′-ATAAGTTCGGCCCCATGTATAAGCGG-3′)，Sf-1-GSP3′(5′-CTTTGGCCTCATGTGCAAGATGGCGGAC-3′)和Sf-1-NP3′(5′-TGCTGGTGTTTGACCACATCTACCG-3′)，特异性引物Sf-1-GSP5与Sf-1-GSP3的核苷酸序列分别如SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9、SEQ ID NO：10所示。
按照Smart RACE试剂盒说明书进行5′RACE和3′RACE扩增，获得中华鳖Sf-1基因cDNA片段的5′末端和3′末端。将中华鳖Sf-1基因的5′末端和3′末端进行拼接，获得其cDNA全长序列。中华鳖Sf-1基因的核苷酸和氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示。
5′RACE-PCR扩增第一轮反应体系为(20μl)：
| 组成 | 体积(μL) |
| Premix Taq<sup>TM</sup>(Ex Taq<sup>TM</sup> Version 2.0plus dye) | 16.6 |
| 5′-RACE-Ready cDNA | 1.0 |
| Sf-1-GSP5′ | 0.4 |
| μPM-Long | 2.0 |
5′RACE-PCR扩增第二轮反应体系为(10μL)(以第一轮PCR产物为反应模板)：
组成体积(μL)
3′RACE-PCR扩增的反应体系为(20μL)：
5′RACE-PCR扩增第二轮反应体系为(10μL)(以第一轮PCR产物为反应模板)：
反应条件为：
步骤2中华鳖Sf-1基因重组表达载体的构建：
根据中华鳖Sf-1基因的cDNA序列，设计一对特异性引物Sf-1-F1(5′-CGCGGATCCATGACGCCCGTCGACTACGAC-3′)和Sf-1-R1(5′-CCGCTCGAGCGCCAGTATCCG AGCCTTGA-3′)，分别在上、下游引物添加了BamHⅠ和XhoⅠ酶切位点。以中华鳖cDNA为模板，采用引物Sf-1-F1和Sf-1-R扩增中华鳖Sf-1基因的成熟肽片段。特异性引物Sf-1-F1和Sf-1-R核苷酸序列分别如SEQ ID NO:3和SEQ ID NO：4所示。
PCR反应体系(10μL)：
反应条件为：
将PCR产物与重组表达载体pET-32a同时进行BamHⅠ和XhoⅠ双酶切，分别用DNA凝胶回收试剂盒纯化酶切产物，经T4DNA连接酶连接后转化大肠杆菌DH5α，在含有氨苄抗性(100mg/ml)的平板上进行抗性筛选。挑取阳性克隆接种到LB液体培养基中进行培养，37℃、220r/min，培养8h后用质粒提取试剂盒提取质粒，采用质粒PCR和双酶切的方法验证重组质粒，检测结果见图1和图2，其中M：DL 2000plus DNA ladder；1：抗原片段电泳结果；2：重组质粒；3：重组质粒单酶切；4：重组质粒双酶切。已成功构建重组表达载体，命名为pET-Sf-1。
步骤3中华鳖Sf-1重组蛋白的表达和纯化：
中华鳖Sf-1重组蛋白的诱导表达：取1ng重组表达载体pET-Sf-1转化大肠杆菌BL21(DE3)涂布于含100mg/ml的氨苄抗性平板上进行抗性筛选，培养温度为37℃，培养时间为过夜培养。筛选结束后转至有氨苄抗性的LB培养基(100mg/ml)中培养，培养条件为37℃，200r/min。菌体进入指数生长期后添加IPTG至终浓度1mmol/L，进行IPTG诱导培养4小时后离心收集菌体沉淀，PBS重悬，超声波破碎，加入5×上扬缓冲液进行常规SDS-PAGE电泳检测重组蛋白的表达情况，重组蛋白分子量为29.9kDa。
中华鳖Sf-1重组蛋白的纯化：将上述未经过IPTG诱导的菌液按照1:100的比例接种到500ml有氨苄抗性的LB培养基(100mg/ml)中进行扩大培养，培养条件为37℃，200r/min至OD值为0.6时，加入IPTG进行诱导，培养4h。培养结束后，离心收集菌体沉淀，用变性结合缓冲液悬浮菌液，进行超声波破碎并收集上清部分。对上清液进行过滤；滤液用1mL的亲和层析镍柱His-Bing-Resin进行纯化；纯化后的蛋白用不同浓度的盐酸胍缓冲液进行透析复性，蛋白用BCA法测蛋白浓度，并用常规SDS-PAGE法，检测重组蛋白的条带大小，检测结果如图3所示。结果表明中华鳖Sf-1重组蛋白为单一条带，分子量为29.9kDa，其中M为低质量蛋白Mark；1为未诱导的pET-32α菌体蛋白；2为未诱导的菌体总蛋白；3为诱导后的菌体总蛋白；4为纯化后蛋白。
中华鳖Sf-1重组蛋白的特异性检测：
取纯化后的重组蛋白30mg，进行SDS-PAGE电泳，然后将蛋白转至PVDF膜上。以带his标签的鼠抗作为一抗，以HRP标记的羊抗鼠IgG为二抗进行蛋白特异性检测。检测结果如图4所示，条带单一且分子量大小一致，为29.9kDa。
步骤4中华鳖Sf-1重组蛋白多克隆抗体的制备：
将纯化的中华鳖Sf-1重组蛋白和等体积的弗氏完全佐剂进行充分乳化；用腹部皮下多点注射法，按0.1mg的免疫量对小鼠进行免疫，采集保存第一次免疫前小鼠血清。用经弗氏不完全佐剂充分乳化的抗原进行加强免疫，每次间隔7天，共注射5次。末次免疫10天后，进行全血分离，收集鼠抗中华鳖Sf-1血清，加入30％甘油，于-80℃分装保存。
步骤5中华鳖Sf-1蛋白的免疫组化分析：
采用石蜡包埋法包埋中华鳖精巢组织，以5μm厚度进行切片。常规脱蜡后，用50μl的H2O2(3％)于37℃孵育10min，阻断内源过氧化物酶。将切片放入柠檬酸盐缓冲液中并加热至沸腾，20min后自然冷却。用5％牛血清蛋白进行封闭后加入100倍稀释的鼠抗中华鳖Sf-1血清37℃温育2h，用PBS洗涤3次，加入HRP标记的山羊抗鼠IgG为二抗，37℃孵育30min，DAB显色。苏木精复染、脱水透明及树胶封片。阴性对照以实施例3中，多克隆抗体的制备步骤中的第一次免疫前鼠血清代替一抗。分析结果如图5和6所示，(a)为阴性对照结果、(b)为免疫组化结果。由免疫组化分析结果可知，中华鳖Sf-1蛋白在间质细胞、精母细胞、精子细胞及胞质中表达。
步骤6多克隆抗体的效价测定：
为了对所获得的多克隆抗体的效价进行测定，具体过程如下：
以实施例4中，多克隆抗体的制备步骤中的第一次免疫前鼠血清为隐性对照，以最后一次免疫后收集的鼠抗中华鳖Sf-1血清在1:100至1:2430000倍之间稀释作为一抗，以HRP标记的羊抗鼠IgG为二抗，以TMB作为显色液，以鼠抗中华鳖Sf-1血清样品OD 450nm值与阴性对照血清OD 450nm值的比值≥2时作为判定标准，以制备的鼠抗中华鳖Sf-1血清最大稀释度作为该抗体的有效效价。检测结果如图7所示，阳性血清的最高稀释度为1:90000，比较对象为阴性血清与鼠抗血清。
上述结果表明，本发明与现有技术相比具有如下有益效果：
1)本发明采用分子生物学和基因工程的方法，构建了中华鳖Sf-1基因的重组表达载体，诱导表达，亲和层析获得纯化的重组表达蛋白。
2)本发明的方法制备的中华鳖Sf-1多克隆抗体具有很强的特异性，可用于免疫组化、免疫印记等实验要求，建立体外免疫分析，为深入研究中华鳖Sf-1的功能提供一个重要工具。
3)本发明制备的中华鳖Sf-1多克隆抗体，可用于中华鳖免疫应该染色激光共聚焦显微观察。
虽然，上文中已经用一般性说明及具体实施例对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 河南师范大学
<120> 中华鳖类固醇生成因子-1多克隆抗体的制备方法及应用
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taagcgggat cgtgccttaa agcagcagaa gaaagctctg atccgcgcca acggctttaa 420
gctggagacg gtgccccaga tcgtgtcccc ggtgcaaacc gactacagcc tctcctccgc 480
catccacggc atccactctg tggccaagag cctgccgccc aacccggccg ctatgacgcc 540
cgtcgactac gacaggagcc cctacgggac gccgtccctg ggcatgactg tgccgagcca 600
cggggccctg tccagctacc actaccctgc ctttcccaac cgcaccatca agtccgagta 660
ccccgaccac tacacaagtt cccacgagtc cgtggccagc tacatgtatc ccgacgcctg 720
ccccagcagc tccccgcccg gcatccccga ggtcattctc aagctgctgc agctggagcc 780
ggacgagccg caggtcaagg ctcggatact ggcgtgcctg caacaggagc agggcaaagg 840
ccggcatgag aagctccaca cctttggcct catgtgcaag atggcggacc agaccctctt 900
ctccatcgtg gagtgggcac ggagctgtgt cttcttcaag gagctggagg tgggcgacca 960
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ggtgcagcac gggaaggagc acagcatgct gcttgtgacg ggccaggagg tggacatggc 1080
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ggtgctgcac ctgcagtcgc tccaggtgga cagacaagag tttgtgtgtc tgaagttcct 1200
gatcctcttc agcctggacg tgaagtacct ggagaaccac agcctggcga aggcggcgca 1260
ggagaaggcc aacgcggccc tgctggagta cacggcctgc cactacccgc actgcgggga 1320
caagtgccgc cagctcctgc tgcggctggc cgaggtgcgc gccctcagca tgcaggcgga 1380
ggagtacctc taccacaagc acctgagcgg ggacgtgccg tgcaacaacc tgctcatcga 1440
gatgctgcac gccaagcgga cttgagcgcg gcccagggac acgccacgga gcatctggcc 1500
cccccgcggg gagcgagtgg gtcaggcccc gggggagggg ggtctggcca cgcagccctg 1560
cccgcccggc gccgctcgcc ggggcgtgtt tcaccagaac tgttggccgg gcgcagatga 1620
tctattttaa tacacaggaa cttcgtttca cggctaaaaa aaaaaaaaaa aaaaaaaaaa 1680
aa 1682
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Met Thr Val Pro Ser His Gly Ala Leu Ser Ser Tyr His Tyr Pro Ala
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Glu Pro Asp Glu Pro Gln Val Lys Ala Arg Ile Leu Ala Cys Leu Gln
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Gln Glu Gln Gly Lys Gly Arg His Glu Lys Leu His Thr Phe Gly Leu
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Arg Gln Val Gln His Gly Lys Glu His Ser Met Leu Leu Val Thr Gly
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Gln Glu Val Asp Met Ala Thr Val Ala Ala Gln Ala Gly Ser Ile Met
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Phe Ser Leu Asp Val Lys Tyr Leu Glu Asn His Ser Leu Ala Lys Ala
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Claims (10)
1.一种中华鳖类固醇生成因子-1多克隆抗体的制备方法，其特征在于，包括以下步骤：
步骤一、中华鳖Sf-1基因的克隆：
提取中华鳖总RNA，用M-MLV反转录酶合成cDNA第一链，于温度为-80℃保存；根据GenBank数据库中的Sf-1基因序列，设计第一对特异性引物，进行PCR扩增，获得中华鳖Sf-1基因cDNA片段；进行5′RACE-PCR和3′RACE-PCR扩增，获得中华鳖Sf-1基因cDNA片段的5′末端和3′末端，拼接获得中华鳖Sf-1基因全长cNDA序列；中华鳖Sf-1基因的核苷酸和氨基酸序列分别如SEQ ID NO：1和SEQ ID NO：2所示；
步骤二、中华鳖Sf-1基因重组表达载体的构建：
构建中华鳖Sf-1基因全长cDNA序列，设计第二对特异性引物Sf-1-F1和Sf-1-R1，分别在上、下游引物添加BamH Ⅰ和Xho Ⅰ酶切位点；以中华鳖cNDA为模板，Sf-1-F1和Sf-1-R1为引物，进行PCR扩增；将PCR产物和pET-32a(+)表达载体进行双酶切，回收纯化，连接转化大肠杆菌DH5α，获得重组表达载体pET-Sf-1；
步骤三、中华鳖Sf-1重组蛋白的诱导表达和纯化及Western Blot进行特异性分析：
将步骤二得到的重组表达载体pET-Sf-1转化大肠杆菌BL21(DE3)，在含有100mg/mL氨苄抗性的LB培养液中培养至OD值为0.6时，加入IPTG进行诱导；经离心收集菌体、PBS重悬和超声波破碎后，加入5×上样缓冲液进行12％SDS-PAGE电泳检测目的蛋白的表达情况；
以1％的接种量将能表达重组蛋白的BL21添加到含有100mg/mL氨苄抗性的LB培养基中，大量培养，诱导表达；将诱导好的菌液离心，收集菌体用变性结合缓冲液溶解，超声波破碎，再离心收集上清，上样、结合、洗脱、透析，获得纯化的中华鳖Sf-1重组蛋白；SDS-PAGE电泳检测纯化的中华鳖Sf-1重组蛋白；
步骤四、多克隆抗体的制备：
将步骤三纯化的pET-Sf-1重组蛋白作为抗原，对小鼠进行免疫，按体积比1:1的比例加入弗氏完全佐剂，采用腹部皮下多点注射；加强免疫选用弗氏不完全佐剂；末次免疫10天后，进行全血分离，收集鼠抗血清，纯化得到中华鳖Sf-1多克隆抗体。
2.根据权利要求1所述中华鳖类固醇生成因子-1多克隆抗体的制备方法，其特征在于，步骤一中第一对特异性引物为：
上游引物名称为Sf-1 F，如SEQ ID NO:3所示；
引物序列为5′-GCTGCCACCGTACCCATACTCCT-3′；
下游引物名称为Sf-1R，如SEQ ID NO:4所示；
引物序列为5′-AGTAAGGGCTGTCTTTCCTTCG-3′。
3.根据权利要求1所述中华鳖类固醇生成因子-1多克隆抗体的制备方法，其特征在于：步骤一中设计第一对特异性引物进行PCR扩增时，每10μL的PCR扩增的反应体系为包括ddH2O 2.4μL、Premix TaqTM 5.0μL、Sf-1-F 0.3μL、Sf-1-R 0.3μL、cDNA 2.0μL。
4.根据权利要求3所述中华鳖类固醇生成因子-1多克隆抗体的制备方法，其特征在于：步骤一中设计第一对特异性引物进行PCR扩增时，反应条件为：预变性94℃持续3min，变性94℃持续30s，退火56℃持续30s，延伸72℃持续1.5min，最后延伸72℃持续10min，期间，变性、退火、延伸进行30次循环。
5.根据权利要求1所述中华鳖类固醇生成因子-1多克隆抗体的制备方法，其特征在于，步骤二中的第二对特异性引物为：
上游引物名称为Sf-1-F1，如SEQ ID NO:5所示；
引物序列为：5′-CGCGGATCCATGACGCCCGTCGACTACGAC-3′；
下游引物名称为Sf-1-R1，如SEQ ID NO:6所示；
引物序列为：5′-CCGCTCGAGCGCCAGTATCCGAGCCTTGA-3′。
6.根据权利要求1所述中华鳖类固醇生成因子-1多克隆抗体的制备方法，其特征在于：步骤二中设计第二对特异性引物进行PCR扩增时，每10μL的PCR扩增的反应体系为包括ddH2O 2.4μL、Premix TaqTM 5.0μL、Sf-1-F1 0.3μL、Sf-1-R1 0.3μL、cDNA 2.0μL。
7.根据权利要求6所述中华鳖类固醇生成因子-1多克隆抗体的制备方法，其特征在于：步骤一中设计第一对特异性引物进行PCR扩增时，反应条件为：预变性94℃持续3min、变性94℃持续30s、退火60℃持续30s、延伸72℃持续30s、最后延伸72℃持续10min，期间，变性、退火、延伸进行35次循环。
8.根据权利要求1所述中华鳖类固醇生成因子-1多克隆抗体的制备方法，其特征在于：步骤三中，加入IPTG进行诱导时，IPTG的添加时间点为大肠杆菌BL21(DE3)进入指数生长期时，IPTG的终浓度为1mmol/L，IPTG的诱导时间为4小时；所述变性结合缓冲液分为20mM磷酸钠，15mM咪唑，500mM氯化钠，6M尿素，pH调整为7.4。
9.根据权利要求1所述中华鳖类固醇生成因子-1多克隆抗体的制备方法，其特征在于：步骤三中中华鳖Sf-1重组蛋白分子量为29.9kDa，步骤四中免疫过程中免疫量为0.1mg。
10.根据权利要求1—9中任一项所述方法制备的中华鳖类固醇生成因子-1多克隆抗体于中华鳖免疫荧光染色激光共聚焦显微观察中的应用。
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