CN107936107B - 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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- CN107936107B CN107936107B CN201711420022.3A CN201711420022A CN107936107B CN 107936107 B CN107936107 B CN 107936107B CN 201711420022 A CN201711420022 A CN 201711420022A CN 107936107 B CN107936107 B CN 107936107B
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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；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；
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SEQ IDNO.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；
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 μL TBS（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。
实验例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，采用引物P3和P4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，采用引物P5和P6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，500ul/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/CgIFNLP promoter + 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；
（8）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
<110>大连海洋大学
<120>长牡蛎干扰素调节因子CgIRF-1基因重组蛋白
<160>1
<170>SIP0SequenceListing 1.0
<210>1
<211>329
<212>PRT
<213>长牡蛎（Crassostrea gigas）
<400>1
Met Gly Ser Asp Lys Met Lys Arg Ser Asp Glu Lys Met Thr Lys Lys
Arg Pro Val Glu Arg Gln Lys Met Arg Pro Trp Leu Met Asp Met Leu
Lys Gln Gly Ala Thr Gln Gly Leu Glu Trp Phe Asp Glu Thr Gln Lys
Leu Phe Lys Ile Asn Trp Lys His Gly Ser Arg His Gly Phe Asn Thr
Met Lys Asp Ala Ser Leu Phe Glu Lys Tyr Ala Gln His Thr Gly Arg
Trp Asp Pro Asp Asp Pro Ser Pro Lys Arg Trp Lys Ala Asn Phe Arg
Cys Ala Leu Asn Ser Leu Gln Asn Val Met Glu Ile Lys Lys Leu Gly
Glu Ser Lys Gly Val His Ala Phe Arg Val Tyr Gln Phe Leu Glu Glu
Asp Asp Thr Lys Pro Lys Glu Gly Asn Ala Arg Lys Ser Asn Lys His
Lys Ser Asn Gln Lys Ser Thr Arg Lys Val Asn Ala Lys Phe Val Glu
Arg Glu Arg Glu Glu Ser Asp Lys Glu Asp Cys Pro Leu Asp Asp His
Glu Arg Glu Ile Pro Thr Ser Thr Ser Gly Glu Gly Glu Glu Val Ser
Glu Asn Pro Pro Gln Cys Thr Lys Pro Glu Ile Arg Val Phe Thr Phe
Pro Thr His Met Ala Gly Cys Val Val Arg Met Ala Pro Arg Lys Arg
Leu Ala Pro Tyr Glu Leu Glu Glu Glu Asp Lys Glu Asp Gln Val Glu
Met Thr Ser Gln Glu Thr Ile Gln His Ile Gln Tyr Thr Thr Lys Arg
Arg Arg Arg Asp Thr Asp Asp Glu Ser Met Met Ser Tyr Thr Gln Pro
Ser Ile Thr Thr Asp Glu Ser Ser Asn Asp Ser Asn Ser Ser Val Ser
Ser Ser Ser Val Glu Glu Glu Ile Pro Asn Thr Pro Tyr Phe Ser Ser
Leu Leu Thr Asn Ile Leu Asp Asp Asp Trp Thr Val Glu Lys Glu Glu
Thr Val Thr Thr Thr Gln Gln Tyr Val 329
Claims (1)
1.一种长牡蛎干扰素调节因子CgIRF-1基因重组蛋白在制备诱导CgIFNLP基因表达药物中的应用，其特征在于：所述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。
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