CN107936107A - 长牡蛎干扰素调节因子CgIRF‑1基因重组蛋白、制备方法及应用 - Google Patents
长牡蛎干扰素调节因子CgIRF‑1基因重组蛋白、制备方法及应用 Download PDFInfo
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- CN107936107A CN107936107A CN201711420022.3A CN201711420022A CN107936107A CN 107936107 A CN107936107 A CN 107936107A CN 201711420022 A CN201711420022 A CN 201711420022A CN 107936107 A CN107936107 A CN 107936107A
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Abstract
本发明公开一种长牡蛎干扰素调节因子CgIRF‑1基因重组蛋白，氨基酸序列如SEQ ID NO.1所示。制备方法依次按照如下步骤进行：用特定引物P1和P2对长牡蛎干扰素调节因子CgIRF‑1基因编码区片段进行PCR扩增，所述引物P1的DNA序列如SEQ ID NO.2所示，所述引物P2的DNA序列如SEQ ID NO.3所示；将PCR扩增产物与pET‑30a载体经Nde I、Xho I酶切后通过连接酶连接，转化，测序鉴定重组子；将上述重组子转入大肠杆菌BL21（DE3）表达菌株中进行诱导培养，而后纯化、复性即可。所述长牡蛎干扰素调节因子CgIRF‑1基因重组蛋白可应用于制备免疫增强剂。
Description
技术领域
本发明属于分子生物学技术领域，尤其涉及一种长牡蛎干扰素调节因子CgIRF-1基因重组蛋白、制备方法及应用。
背景技术
长牡蛎是一种重要的海水养殖贝类。由于长牡蛎缺乏适应性免疫防御系统，主要依赖固有免疫系统抵御外源病原微生物的侵染，因此细菌、真菌和病毒引起的多种疾病在长牡蛎的养殖群体中不断爆发，造成了巨大的经济损失。
干扰素（IFN）是一类能够抵抗病毒感染，抑制细胞周期和行使免疫调节功能的细胞因子。干扰素调节因子（IRFs）是调节干扰素（IFN）、干扰素刺激基因（ISG）以及其它相关基因表达的重要转录因子，能够与干扰素（IFN）或者干扰素刺激基因（ISG）上游启动子区含有5’-GAAA-3’保守基序的DNA序列相结合，并通过调节IFN、ISG和其它密切相关基因的表达而发挥多种生物学效应。在脊椎动物中，已经鉴定出了11个IRF，分别是IRF-1 至IRF-11。所有的IRFs成员在其N端都含有一个保守的螺旋-转角-螺旋型的IRF结构域，又称为DNA锚定结构域。在这个结构域中含有5个保守的色氨酸残基（Trp），这5个保守的残基对识别含有5’-GAAA-3’四核苷酸的DNA序列具有重要作用。IRFs成员IRF-1和IRF-2的C端分别含有一个保守性很低的激活结构域和抑制结构域。研究发现，IRF-1与IRF-2不仅参与病毒防御，还在细胞增殖、分化、凋亡等细胞反应以及很多疾病的调节等发面发挥着重要作用。
但是，迄今为止并没有关于长牡蛎干扰素调节因子CgIRF-1基因重组蛋白、制备方法及在制备免疫增强剂中应用的相关报道。
发明内容
本发明是为了解决现有技术所存在的上述技术问题，提供一种长牡蛎干扰素调节因子CgIRF-1基因重组蛋白、制备方法及应用。
本发明的技术解决方案是：一种长牡蛎干扰素调节因子CgIRF-1基因重组蛋白，其特征在于：氨基酸序列如SEQ ID NO.1所示。
一种上述长牡蛎干扰素调节因子CgIRF-1基因重组蛋白的制备方法，其特征在于依次按照如下步骤进行：
a. 用特定引物P1和P2对长牡蛎干扰素调节因子CgIRF-1基因编码区片段进行PCR扩增，所述引物P1的DNA序列如SEQ ID NO.2所示，所述引物P2的DNA序列如SEQ ID NO.3所示；
b. 将PCR扩增产物与pET-30a载体经Nde I、Xho I酶切后通过连接酶连接，转化，测序鉴定重组子；
c. 将上述重组子转入大肠杆菌BL21（DE3）表达菌株中进行诱导培养，而后纯化、复性，即得到具有序列表SEQ ID NO.1中氨基酸序列的重组蛋白。
一种上述长牡蛎干扰素调节因子CgIRF-1基因重组蛋白在制备免疫增强剂的应用。
本发明的长牡蛎干扰素调节因子CgIRF-1基因重组蛋白能够在体外和IFN刺激反应元件（ISRE）结合，并且能够激活牡蛎中的类干扰素蛋白（IFNLP）的启动子，可作为免疫增强剂，在制备抗病毒类药物、抗癌剂及抗炎剂等方面具有应用价值。
附图说明
图1 为本发明实施例长牡蛎干扰素调节因子CgIRF-1基因重组蛋白诱导和纯化的电泳图。
图2 为本发明实施例长牡蛎干扰素调节因子CgIRF-1基因重组蛋白与ISRE体外结合活性效果图。
图3 为本发明实施例长牡蛎干扰素调节因子CgIRF-1基因重组蛋白在HEK293T细胞中激活CgIFNLP启动子区的功能效果图。
具体实施方式
本发明的长牡蛎干扰素调节因子CgIRF-1基因重组蛋白，氨基酸序列如SEQIDNO.1所示。
上述长牡蛎干扰素调节因子CgIRF-1基因重组蛋白的制备方法，依次按照如下步骤进行：
1. 重组载体的构建
用特定引物P1和P2对长牡蛎干扰素调节因子CgIRF-1基因编码区片段进行PCR扩增，所述引物P1的DNA序列如SEQ ID NO.2所示，所述引物P2的DNA序列如SEQ ID NO.3所示；
PCR反应条件为：首先95 °C预变性5分钟，然后进入下列循环：95 °C变性30秒，55 °C退火30秒，72 °C延伸1 min，共进行30个循环，最后72 °C延伸10分钟。将扩增片段纯化回收，与pMD19-T载体连接。转化后筛选阳性克隆，提取质粒；使用Nde1和Xho1两个酶酶切质粒，用胶回收纯化试剂盒（大连宝生物工程有限公司）对酶切产生的目的片段纯化回收；回收目的片段与经Nde1和Xho1两个酶酶切的表达载体pET-30a连接，完成载体的构建。
2. 重组蛋白rCgIRF-1的表达
以大肠杆菌BL21（(DE3）为重组表达菌株，将上述测序后读码框正确的重组载体转化到表达宿主菌中。挑取单克隆，接种于5 ml 含有卡那抗生素的LB液体培养基中，37 °C摇床中震荡培养12~16 h，然后以1：100的比例接种200 mL 含有卡那抗生素的LB液体培养基中，37°C，200 rpm，培养至OD600＝0.5~0.7。加入IPTG，使终浓度达到1 mM mL-1，130 rpm，16 °C继续培养4小时。4 °C，10000 rpm，离心10 min，收集菌体，于-20 °C冻存备用。取5 mL菌液离心，弃去上清后，加入800 μL TBS重悬，超声破碎后吸出上清备用，破碎后的沉淀加入80 μLTBS（20 mmol L-1 Tris-HCl, 150 mmol L-1 NaCl, pH 8.0）彻底重悬。分别取20 μL破碎后的菌液上清和重悬后的沉淀加入20 μL 2倍的蛋白上样缓冲液，100 °C煮沸10 min，稍离心，SDS-PAGE检测表达产物。
3. 重组蛋白rCgIRF-1的纯化
采用镍琼脂糖凝胶FF柱纯化获得了可溶的重组蛋白rCgIRF-1，具体操作步骤如下：
（1）镍琼脂糖凝胶 FF装柱，1.6×20cm，柱床体积为10 mL；
（2）用缓冲液I，即TBS平衡2~5个床体积，流速为2 mL min-1；
（3）经IPTG诱导表达的细胞，用缓冲液I重悬，150瓦超声破碎30分钟，12000 rpm，
4 °C离心30 min，上清液用0.45 μm 滤膜过滤后，过柱，流速为1 mL min-1；
（4）用缓冲液1再洗2~5个床体积，流速为2 mL min-1；
（5）用有20 mmol L-1咪唑的缓冲液I再洗2~5个柱床体积，流速为2 mL min-1；
（6）用300 mmol L-1咪唑的缓冲液I洗脱目的蛋白，收集；
（7）用SDS-PAGE检测融合蛋白的表达；
（8）用纯水流洗5个柱床体积，再用20%的乙醇流洗3个柱床体积，流速为2 mL min-1，柱子置于4 °C环境中保存。非变性状态下提纯的重组蛋白需要在TBS缓冲液及纯水中分别透析3次除去咪唑。每次在4 °C透析12 h，即得到长牡蛎干扰素调节因子CgIRF-1基因重组蛋白，氨基酸序列如SEQ ID NO.1所示。
长度：329个氨基酸
类型：氨基酸
链型：单链
特性：分子量为38.383 kDa，等电点为5.67，具有一个保守的IRF结构域即DNA锚定结构域。
本发明实施例长牡蛎干扰素调节因子CgIRF-1基因重组蛋白诱导和纯化的电泳如图1所示。
图1中泳道M：蛋白marker；1：未诱导的重组蛋白rCgIRF-1菌液；2：重组蛋白rCgIRF-1菌液诱导后破碎沉淀；3：重组蛋白rCgIRF-1菌液诱导后破碎上清；4：纯化后的重组蛋白rCgIRF-1。
实验例1：本发明的长牡蛎干扰素调节因子CgIRF-1基因重组蛋白与ISRE的体外结合活性检测
具体步骤如下：
（1）未经生物素标记的合成
ISRE（ISRE-WT (forward)：TGCAGGGAAACTGAAACTAAT；
ISRE-WT(reverse)：ATTAGTTTCAGTTTCCCTGCA），
生物素标记的ISRE（ISRE-Bio (forward)：
biotin-TGCAGGGAAACTGAAACTAAT；
ISRE-Bio (reverse)：biotin-ATTAGTTTCAGTTTCCCTGCA）；
未经生物素标记的突变的ISRE（ISRE-MU (forward)：
TGCAGGCAAACTCAAACTAAT；
ISRE-MU (reverse)：ATTAGTTTGAGTTTGCCTGCA）寡聚核苷酸链。
（2）用退火缓冲液将未经生物素标记的合成ISRE及未经生物素标记的突变的ISRE溶解稀释至100 μM，生物素标记的ISRE溶解稀释至10 μM后取等量的正反链探针退火（95 °C，5 min，缓慢降温至室温）形成双链。
（3）设置5个实验组分别为阴性对照组（重组蛋白rCgIRF-1量为0 μg），阳性对照组（r重组蛋白CgIRF-1量为5 μg），冷竞争探针组（加入终浓度为20 μM 的ISRE-WT竞争探针及5μg重组蛋白CgIRF-1），冷竞争突变探针组（加入终浓度为20 μM 的ISRE-MU竞争探针及5 μg重组蛋白rCgIRF-1）以及超迁移组（加入2 μL CgIRF-1抗体及5μg重组蛋白rCgIRF-1），根据化学发光EMSA试剂盒（碧云天生物技术有限公司）的说明书进行后续的实验，验证本发明的长牡蛎干扰素调节因子CgIRF-1基因重组蛋白与ISRE的体外结合活性。
实验结果如图2所示。结果表明：当未加入重组蛋白rCgIRF-1时，没有出现结合条带；加入重组蛋白rCgIRF-1后，形成结合条带；当加入竞争探针ISRE-WT时，结合条带变弱，而加入关键结合位点突变的ISRE-MU探针时，条带不随着突变探针加入而改变，以上结果说明重组蛋白rCgIRF-1能够在体外与ISRE结合。
实施例2：本发明的长牡蛎干扰素调节因子CgIRF-1基因重组蛋白在HEK293T细胞中促进CgIFNLP启动子区的转录活性分析。
（1）以长牡蛎干扰素调节因子1（CgIRF-1）基因编码区为模板，采用特定引物P3（SEQ ID NO.4）和P4 （SEQ ID NO.5）进行扩增，待用；
（2）采用Kpn I 和 Xho I 对上述的PCR产物及pcDNA3.1 (+)质粒进行酶切，酶切片段通过T4连接酶连接， 转化，测序鉴定重组子；
（3）以长牡蛎类干扰素蛋白（CgIFNLP）上游1000 bp基因非编码区为模板，采用特定引物P5（ SEQ ID NO.6）和P6（ SEQ ID NO.7）进行扩增,待用；
（4）采用Kpn I 和 Xho I 对上述的PCR产物及pGL3 basic进行酶切，酶切片段通过T4连接, 转化，测序鉴定重组子；
（5）将所有的质粒，包括pGL3-control，pGL3-Lu，pcDNA3.1(+)，pRL-TK，pcDNA3.1/CgIRF-1以及pGL3/CgIFNLP promotor进行去内毒素质粒提取；
（6）人肾上皮细胞HEK293T培养于含10％(体积比)胎牛血清的DMEM培养基中，培养条件为置37 °C，5％CO2的培养箱内培养，取对数期细胞用于试验；
（7）将上述细胞分别以0.5×105-2×105/孔的密度接种于24孔细胞培养板，500 ul/孔，置5％CO2培养箱内培养24 h，待细胞贴壁后，更换各自的培养基；
（8）设置三个组分别是阳性对照组：共转染pGL3-control+pRL-TK；空白对照组：分别共转染pGL3-Lu + pcDNA3.1 (+) + pRL-TK和pGL3-Lu + pcDNA3.1/ CgIRF-1 + pRL-TK；实验组：分别共转染pGL3/CgIFNLP promoter + pcDNA3.1 (+) + pRL-TK和pGL3/CgIFNLPpromoter + pcDNA3.1/rCgIRF-1 + pRL-TK。，每个组设6个复孔，每个孔每种质粒总量为0.2 ng，内参质粒pRL-TK总量为0.02 ng。当细胞融合度到达70%-90%时，按照组别并根据LipofectamineTM3000 Reagent说明书进行转染实验；
（9）置5％CO2培养箱内培养48h后用双荧光素报告基因检测试剂盒检测相对荧光活性。
结果如图3所示。图3中pGL-control：共转染pGL-control + pRL-TK；blank：分别共转染pGL3-Lu + pcDNA3.1(+) + pRL-TK和pGL3-Lu + pcDNA3.1/CgIRF-1 + pRL-TK；pGL3/CgIFNLP promoter：分别共转染pGL3/CgIFNLP promoter + pcDNA3.1(+) + pRL-TK和pGL3/CgIFNLP promoter + pcDNA3.1/CgIRF-1 + pRL-TK；其中pRL-TK作为内参质粒。
结果表明：在阳性对照组中，相对荧光活性较高大约是37.59。在空白对照组中，转入空白报告基因质粒pGL3-Lu并分别转入空白表达基因质粒pcDNA3.1 (+)和重组表达基因质粒pcDNA3.1/CgIRF-1时，相对荧光活性分别是0.26 和0.21。在实验组中，转入重组报告基因质粒并分别转入空白表达基因质粒pcDNA3.1 (+)和重组表达基因质粒pcDNA3.1/CgIRF-1时，相对荧光活性分别是3.12 和 6.62，转入pcDNA3.1/CgIRF-1后相对荧光活性增加了2.12倍。由此可见，CgIRF-1在HEK291T细胞中能够诱导CgIFNLP基因的表达。
序列表
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Claims (3)
1.一种长牡蛎干扰素调节因子CgIRF-1基因重组蛋白，其特征在于：氨基酸序列如SEQID NO.1所示。
2.一种如权利要求1所述长牡蛎干扰素调节因子CgIRF-1基因重组蛋白的制备方法，其特征在于依次按照如下步骤进行：
a. 用特定引物P1和P2对长牡蛎干扰素调节因子CgIRF-1基因编码区片段进行PCR扩增，所述引物P1的DNA序列如SEQ ID NO.2所示，所述引物P2的DNA序列如SEQ ID NO.3所示；
b. 将PCR扩增产物与pET-30a载体经Nde I、Xho I酶切后通过连接酶连接，转化，测序鉴定重组子；
c. 将上述重组子转入大肠杆菌BL21（DE3）表达菌株中进行诱导培养，而后纯化、复性，即得到具有序列表SEQ ID NO.1中氨基酸序列的重组蛋白。
3.一种如权利要求1所述长牡蛎干扰素调节因子CgIRF-1基因重组蛋白在制备免疫增强剂的应用。
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| CN109705204A (zh) * | 2019-01-25 | 2019-05-03 | 广东省生物资源应用研究所 | 一种南美白对虾干扰素调节因子及其编码基因 |
| CN114044816A (zh) * | 2021-11-10 | 2022-02-15 | 大连海洋大学 | 重组长牡蛎焦孔素蛋白rCgGSDME-N、制备方法及其应用 |
| CN114044816B (zh) * | 2021-11-10 | 2023-06-16 | 大连海洋大学 | 重组长牡蛎焦孔素蛋白rCgGSDME-N、制备方法及其应用 |
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