CN1473932A - 一种重组石斑鱼腺苷酸环化酶激活多肽基因及其表达系统、表达产物、生产方法和应用 - Google Patents
一种重组石斑鱼腺苷酸环化酶激活多肽基因及其表达系统、表达产物、生产方法和应用 Download PDFInfo
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- CN1473932A CN1473932A CNA031396356A CN03139635A CN1473932A CN 1473932 A CN1473932 A CN 1473932A CN A031396356 A CNA031396356 A CN A031396356A CN 03139635 A CN03139635 A CN 03139635A CN 1473932 A CN1473932 A CN 1473932A
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- adenylate cyclase
- pacap
- activating polypeptide
- cyclase activating
- recombinant
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Abstract
本发明公开了一种石斑鱼腺苷酸环化酶激活多肽基因,该基因是以石斑鱼脑组织总RNA为模板,经RT-PCR和RACE方法而得到的具有石斑鱼腺苷酸环化酶激活多肽基因全长cDNA序列的基因片段;本发明还构建了含该基因的载体和重组工程菌株;本发明还涉及了由该基因编码和重组工程菌株表达的石斑鱼腺苷酸环化酶激活多肽重组蛋白,并提供了该重组蛋白的生产方法;利用本发明可以获得来源稳定、成本便宜的石斑鱼腺苷酸环化酶激活多肽重组蛋白,并进一步制备适合海水鱼类使用的生长与繁殖调控剂。
Description
技术领域
本发明属于基因工程技术领域,涉及一种石斑鱼腺苷酸环化酶激活多肽基因及含有该基因的载体和重组工程菌株以及该基因所编码和表达的产物在制备海水鱼类使用的生长与繁殖调控剂中的应用。
背景技术
脑垂体腺苷酸环化酶激活多肽(pituitary adenylate cyclase activatingpolypeptide,简称:PACAP),是38个氨基酸组成的肽类物质。PACAP最初是从绵羊下丘脑抽提物中被发现的,它属于由血管活性肠肽(VIP)、胰高血糖素(glucagon)、生长激素释放因子和分泌素所组成的基因家族。目前,在鱼类中发现PACAP对鲤科鱼类脑垂体激素分泌活动的具有调节作用,并且证明下丘脑产生的PACAP能在脑垂体水平促进生长激素和促性腺激素的释放。但总体来说,鱼类PACAP的结构与功能、受体结合、基因克隆等方面的研究在国内外还处于起步阶段,虽然编码PACAP的基因在其他一些鱼类中已被克隆,但是对于重要的海洋经济鱼类石斑鱼的PACAP基因的克隆和鉴定则仍未见报道。近年来,了解PACAP生理作用机理,已成为鱼类生理研究的热点。由于PACAP在鱼体内含量极微,分离提纯相当困难,难以应用于生产。因此分离和鉴定PACAP基因,为下一步采用DNA重组技术将其克隆到原核或真核细胞中,可为得到大量廉价的重组PACAP奠定基础。
石斑鱼是海洋珊瑚礁鱼类,属鮨科(Serranidae),石斑鱼属(Epinephelus),是一种名贵的海水鱼类,具有极高的经济价值。但目前在石斑鱼人工养殖中还没有一种能有效地调控其生长与繁殖活动的调控剂。而石斑鱼PACAP则可以用于制备这类调控剂,因此采用DNA重组技术来获得大量的石斑鱼PACAP具有很好的应用前景。
发明内容
本发明的目的在于提供一种重组斜带石斑鱼腺苷酸环化酶激活多肽基因;
本发明的另一目的在于提供含有该基因的载体,特别是克隆载体和表达载体;
本发明的另一目的在于提供上述载体转化的重组工程菌株;
本发明的另一目的在于提供上述基因编码的并由上述重组工程菌株表达的重组斜带石斑鱼腺苷酸环化酶激活多肽;
本发明的另一目的在于提供重组斜带石斑鱼腺苷酸环化酶激活多肽的生产方法;
本发明的另一目的在于提供上述重组多肽在制备海水鱼类使用的生长与繁殖调控剂中的应用。
本发明提供的石斑鱼腺苷酸环化酶激活多肽基因,是以斜带石斑鱼脑组织总RNA为模板,采用RT-PCR和RACE方法而得到的来源于石斑鱼的腺苷酸环化酶激活多肽的成熟肽的结构域所对应的基因片段,其核苷酸序列及其所编码的氨基酸序列如序列表所示。
本发明还构建了含有上述石斑鱼腺苷酸环化酶激活多肽基因的载体,优选的是含有该基因的大肠杆菌表达载体。该载体的构建是按照常规方法,将通过PCR方法扩增合成的石斑鱼腺苷酸环化酶激活多肽基因经酶切和分离纯化后,接入到已有载体的相应酶切位点之间,即构建成所需的含有石斑鱼腺苷酸环化酶激活多肽基因的表达载体。
上述的含有石斑鱼腺苷酸环化酶激活多肽基因的大肠杆菌表达载体优选的是由本发明提供的石斑鱼腺苷酸环化酶激活多肽基因与大肠杆菌载体表达载体pTRX构建而成的重组表达载体,命名pTRX-PACAP。
本发明还用上述含有石斑鱼腺苷酸环化酶激活多肽基因的相应载体进一步构建了高效表达重组石斑鱼腺苷酸环化酶激活多肽的大肠杆菌重组工程菌株。
本发明的能表达重组石斑鱼腺苷酸环化酶激活多肽的大肠杆菌重组工程菌株,优选的是由含有石斑鱼腺苷酸环化酶激活多肽基因的表达载体pTRX-PACAP转化大肠杆菌BL21而得到,并命名为pTRX-PACAP-BL21。
本发明提供了利用该大肠杆菌重组工程菌株生产石斑鱼腺苷酸环化酶激活多肽的方法。先将菌株进行发酵并经IPTG诱导使其高效表达石斑鱼腺苷酸环化酶激活多肽重组蛋白,再收集菌体经分离纯化得到重组石斑鱼腺苷酸环化酶激活多肽产品。
利用该大肠杆菌重组菌株pTRX-PACAP-BL21生产石斑鱼腺苷酸环化酶激活多肽的具体步骤为:
(1)菌种的培养发酵和诱导表达:将菌种接种在含氨苄青霉素的LB丰富液体培养基中,37℃,150-200转/分培养过夜,次日以1∶50转种于相同培养基中,37℃,150-200转/分培养至OD600为0.5,加IPTG至终浓度为0.5mol/L,;25℃继续振荡培养13小时后收菌。
(2)表达产物的纯化:收集上述纯化的菌体,将细胞破碎后离心收集包含体沉淀,菌体超声裂解液进行Ni-Chelsting亲和层析,Sephadex G-25凝胶过滤脱盐、去咪唑,DEAE Sepharose阴离子交换柱浓缩并去除碱性杂蛋白,Sephadex G-25凝胶过滤脱盐,25℃肠激酶切割PACAP-PTRX融合蛋白。DEAE Sepharose阴离子交换层析出去残余酸性杂蛋白,PACAP-PTRX成熟蛋白经Sephadex G-25分子筛更换PB缓冲系统,SP Sepharose阳离子交换柱浓缩并出去杂蛋白,经冻干即为纯化的重组石斑鱼腺苷酸环化酶激活多肽。
上述重组石斑鱼腺苷酸环化酶激活多肽可以直接用于制备适合海水鱼类尤其是石斑鱼属使用的生长与繁殖调控剂,也可以通过适当的赋型剂、保护剂按一定比例形成组合物来应用。
本发明提供的石斑鱼腺苷酸环化酶激活多肽基因通过与适当的载体连接后可在相应的微生物菌株中高效表达,分离纯化后即可得高纯度的重组石斑鱼腺苷酸环化酶激活多肽,而且具有表达水平高,容易纯化的优点。所以通过本发明,可以方便地大量生产重组石斑鱼腺苷酸环化酶激活多肽,成本较低。这种来源稳定、成本便宜的重组石斑鱼腺苷酸环化酶激活多肽可进一步应用于制备适合海水鱼类尤其是石斑鱼属使用的生长与繁殖调控剂。
附图说明
图1是通过PCR扩增得到石斑鱼腺苷酸环化酶激活多肽(PACAP)cDNA的凝胶电泳分析图。其中1.100bp DNA相对分子质量标准;2.第一轮PCR产物;3.嵌套PCR产物。
图2是3’RACE的结果。其中1.100bp DNA相对分子质量标准;2.第一轮PCR产物;3.嵌套PCR产物。
图3是5’RACE的结果。其中1.100bp DNA相对分子质量标准;2.加尾反应20分钟产物;3.加尾反应5分钟产物。
图4是重组表达载体PACAP-PTRX的构建过程示意图。
图5是重组石斑鱼腺苷酸环化酶激活多肽表达产物的SDS-PAGE凝胶电泳图谱。其中1.蛋白质相对分子质量标准;2.BL21不诱导;3.pTRX不诱导;4.pTRX-PACAP-BL21诱导。
具体实施例实施例1石斑鱼腺苷酸环化酶激活多肽的cDNA的合成
根据已知的哺乳动物和石斑鱼的石斑鱼腺苷酸环化酶激活多肽cDNA序列,设计三条引物:senseP1:5’GCA(GT)G(AG)C(AC)A(AGT)(AG)TCTA GTA(AG)
AG C(GT) ACT 3’senseP2:5’GA(AG)A(AG)GA(AG)C(GC)GAAA(GC)GCATGCAG3’antisenseP3:5’TA(AGC)(AGC)C(ACT)A(AG)(GCT)CG(AGC)CGTCCTTTG3’
senseP1、antisenseP3为合成PACAP 524 bp引物,senseP2、antisenseP3为合成PACAP编码区288bp cDNA的引物。以脑总RNA的反转录产物cDNA为模板经PCR方法扩增合成PACAP 524bp、合成PACAP编码区288bp的基因。PCR反应条件为:变性94℃3分钟;94℃45秒,退火55℃30秒,延伸72℃1分30秒,25个循环;结束后72℃延伸10分钟。
PCR扩增产物的电泳鉴定见图1。从图1可见PCR扩增了一段约290bp的序列。
将扩增产物连接到pGEM-T Easy载体上得到重组质粒,以重组质粒为模板,应用通用引物T7和SP6进行测序,并与其它哺乳动物和石斑鱼进行同源性比较,结果表明克隆的PCR产物是石斑鱼腺苷酸环化酶激活多肽。测序结果见序列表。实施例2含石斑鱼腺苷酸环化酶激活多肽基因的大肠杆菌表达载体pTRX-PACAP的构建
将含有双酶切位点和PACAP序列的PCR产物经过限制性酶KpnI及NotI酶切后进行凝胶电泳,分离纯化约140bp的石斑鱼腺苷酸环化酶激活多肽片段;大肠杆菌表达质粒pTRX经限制性酶KpnI及NotI酶切后分离纯化5.8kb的大片段,与140bp的石斑鱼腺苷酸环化酶激活多肽片段按1∶3混合,用T4连接酶连接16小时后转化入感受态大肠杆菌BL21中,筛选具氨卞青霉素抗性的转化子,用Omega公司质粒提出试剂盒筛选大小约6kb的重组质粒,用限制性酶KpnI及NotI酶切重组质粒,得到140bp和5.8kb的两个片段,大小分别与石斑鱼腺苷酸环化酶激活多肽和表达载体pTRX大小相同,表明石斑鱼腺苷酸环化酶激活多肽已克隆入大肠杆菌表达载体pTRX中,重组质粒命名为pTRX-PACAP(质粒构建图见图4)。实施例3能高效表达重组石斑鱼腺苷酸环化酶激活多肽的大肠杆菌重组工程菌株pTRX-PACAP-BL21的构建
用CaCl2法将pTRX-PACAP转入大肠杆菌BL21,在含氨苄青霉素的LB平板上筛选转化子,经质粒检测和酶切分析获得含pTRX-PACAP的重组转化子pTRX-PACAP-BL21。
实施例4利用大肠杆菌重组工程菌株pTRX-PACAP-BL21生产重组石斑鱼腺苷酸环化酶激活多肽。(1)菌种的培养发酵
将菌种接种在含氨苄青霉素的LB丰富液体培养基中,37℃,150-200转/分培养过夜,次日以1∶50转种于相同培养基中,37℃,150-200转/分培养至OD600为0.5,加IPTG至终浓度为0.5mol/L,;25℃继续振荡培养13小时后收菌。
取少量细胞加2x上样缓冲液,煮沸5分钟后,按标准方法进行SDS-PAGE凝胶电泳。结果见图5,显示经诱导的pTRX-PACAP-BL21在20kd的位置上,出现一条新的蛋白带,经诱导的空载体pTRX及空宿主BL21不出现此带,证明石斑鱼腺苷酸环化酶激活多肽已在pTRX-PACAP-BL21中诱导表达。(2)重组的石斑鱼腺苷酸环化酶激活多肽的纯化
收集上述纯化的菌体,将细胞破碎后离心收集包含体沉淀,菌体超声裂解液进行Ni-Chelsting亲和层析,Sephadex G-25凝胶过滤脱盐、去咪唑,DEAESepharose阴离子交换柱浓缩并去处碱性杂蛋白,Sephadex G-25凝胶过滤脱盐,25℃肠激酶切割pTRX-PACAP融合蛋白。DEAE Sepharose阴离子交换层析出去残余酸性杂蛋白,pTRX-PACAP成熟蛋白经Sephadex G-25分子筛更换PB缓冲系统,SP Sepharose阳离子交换柱浓缩并除去杂蛋白,经冻干即为纯化的重组石斑鱼腺苷酸环化酶激活多肽。
序列表<110>中山大学<120>一种重组石斑鱼腺苷酸环化酶激活多肽基因及其表达系统、表达产物、生产方法和应用<160>2<210>1<211>1028<212>DNA<213>斜带石斑鱼(Epinephelus coioides)<220><221>CDS<222>(339)---(866)<220><221>Sig_peptide<222>339--404<400>1GGGGGGGGGG CAAGGCTTAC GATACACGGA TAAAAATCCA AGGCAATCAC AGCACAGGAC 60AAAAGTTTTG GAGCAACGAT ATGCAGACGA ACATACGGCG CACTTTGCCC AGCCGAGTTT 120CTCTGTTCGC CGGTGCGACT GCTTGACTCC TATTAATTGT GTCTAAGAGT GAGATAGGCT 180GCACTGAGGG GAACATAAGG AGACGACGGA GGCGCTTCAG CCAGGGAAGA AGTGCCGTGA 240GAAAGAAACC GTCTCTCTCA CACACTCACA CACACTTACC ACCACAGCCG CGCCCGGATC 300ACATCCTACA GCACAGCGCT CTGCTCCCCT GCTATAGC ATG GCC AGT TCG AGT AAA 356
Met Ala Ser Ser Ser Lys 6GCC ACT TTA ATC TTG CTC GTC TAC GGA ATC TTA ATG CAC TAC AGC GTC TTC 407Ala Thr Leu Ile Leu Leu Val Tyr Gly Ile Leu Met His Tyr Ser Val Phe 23TGC ACA CCT ATC GGA CTA AGT TAC CCT AAG ATT AGA CTT GAA AAC GAC GCC 458Cys Thr Pro Ile Gly Leu Ser Thr Pro Lys Ile Arg Leu Glu Asn Asp Ala 40TTC GAT GAG GAT GGG AAT GCA TTA TCC GAC ATG GGT TTT GAT AGC GAT CAG 509Phe Asp Glu Asp Gly Asn Ala Leu Ser Asp Met Gly Phe Asp Ser Asp Gln 57ATT GCT ATA CGA AGC CCA CCA TCC TTA AAC GAC GAC GCG TAC ACT CTC TAC 560Ile Ala Ile Arg Ser Pro Pro Ser Leu Asn Asp Asp Ala Tyr Thr Leu Tyr 74
74TAC CCA CCA GAG AAG AGA CCA GAA AGG CAT GCT GAG GAA GAA TTA GAT AGA 611Tyr Pro Pro Glu Lys Arg Pro Glu Arg His Ala Glu Glu Glu Leu Asp Arg 91
91GCC TTG AGG GAG ATC CTG GGT CAG TTA ACA GCG AGA CAT TAT CTG CAT TCT 662Ala Leu Arg Glu Ile Leu Gly Gln Leu Thr Ala Arg His Tyr Leu His Ser 108
108CTG ATG ACA ATT CGT GCA GGT GAA GAC AAC AGC ATG GAG GAA GAG TCA GAG 713Leu Met Thr Ile Arg Ala Gly Glu Asp Asn Ser Met Glu Glu Glu Ser Glu 125CCC TTA TCC AAA AGA CAT TCA GAT GGG ATC TTC ACC GAC AGC TAC AGT CGC 764Pro Leu Ser Lys Arg His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg 142TAT AGA AAG CAG ATG GCC GTG CAG AAA TAC CTG GCA GCG GTT CTG GGA AGA 815Tyr Arg Lys Gln Met Ala Val Gln Lys Tyr Leu Ala Ala Val Leu Gly Arg 159AGG TAC AGA CAG AGA GTT AGG AAC AAA GGA CGC CGG CTT GCC TAT TTG TAG 866Arg Tyr Arg Gln Arg Val Arg Asn Lys Gly Arg Arg Leu Ala Tyr Leu * 175CGTTGTTACA GCGCCCTTAA ACTGCCCTCC TGTGTATATA CATCCAGTCG TTAAATCAAA 926GTCATTCAGA TATATCTGAC CAACCAGTGG ATTGCGCCTG TGTTCTTTCA ACATGTATTT 986ATGTATGAAG TAAAGCCATT AAAATGAATA TTTTAATAATAA 1028<210>2<211>175<212>PRT<213>斜带石斑鱼(EpinePhelus coioides)<400>2Met Ala Ser Ser Ser Lys Ala Thr Leu Ile Leu Leu Val Tyr Gly Ile 16Leu Met His Tyr Ser Val Phe Cys Thr Pro Ile Gly Leu Ser Thr Pro 32Lys Ile Arg Leu Glu Asn Asp Ala Phe Asp Glu Asp Gly Asn Ala Leu 48Ser Asp Met Gly Phe Asp Ser Asp Gln Ile Ala Ile Arg Ser Pro Pro 64Ser Leu Asn Asp Asp Ala Tyr Thr Leu Tyr Tyr Pro Pro Glu Lys Arg 80Pro Glu Arg His Ala Glu Glu Glu Leu Asp Arg Ala Leu Arg Glu Ile 96Leu Gly Gln Leu Thr Ala Arg His Tyr Leu His Ser Leu Met Thr Ile 112Arg Ala Gly Glu Asp Asn Ser Met Glu Glu Glu Ser Glu Pro Leu Ser 128lys Arg His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg 144Lys Gln Met Ala Val Gln Lys Tyr Leu Ala Ala Val Leu Gly Arg Arg 160Tyr Arg Gln Arg Val Arg Ash Lys Gly Arg Arg Leu Ala Tyr Leu * 175
Claims (11)
1、一种石斑鱼腺苷酸环化酶激活多肽基因,其核苷酸序列及其编码的氨基酸序列如序列表所示。
2、如权利要求1所述的石斑鱼腺苷酸环化酶激活多肽基因,其特征在于它是以石斑鱼脑组织总RNA反转录得到的cDNA为模板,经PCR和RACE方法扩增而得到的具有石斑鱼腺苷酸环化酶激活多肽全长cDNA的核苷酸序列。
3、一种克隆载体,其特征在于含有权利要求1所述的石斑鱼腺苷酸环化酶激活多肽基因的。
4、如权利要求3所述的克隆载体,其特征在于该载体为大肠杆菌克隆载体pGEMT-PACAP。
5、一种重组工程菌株,其特征在于是由权利要求4所述的克隆载体pGEMT-PACAP转入大肠杆菌DH5α中所形成的大肠杆菌重组工程菌株pGEMT-PACAP-DH5α。
6、一种表达载体,其特征在于含有权利要求1所述的石斑鱼腺苷酸环化酶激活多肽基因。
7、如权利要求6所述的表达载体,其特征在于该载体为大肠杆菌表达载体pTRX-PACAP。
8、一种重组工程菌株,其特征在于是由权利要求7所述的表达载体pTRX-PACAP转入大肠杆菌BL21(DE3)中所形成的大肠杆菌重组工程菌株pTRX-PACAP-BL21。
9、利用权利要求8所述的大肠杆菌重组工程菌株生产重组石斑鱼腺苷酸环化酶激活多肽的方法,其步骤为:
(1)菌种的培养发酵和诱导表达:将菌种接种在含氨苄青霉素的LB丰富液体培养基中,37℃培养过夜,次日转种于相同培养基中,37℃培养至OD600为0.5,加IPTG至终浓度为0.5mol/L;25℃继续振荡培养13小时后收菌。
(2)表达产物的纯化:收集菌体,细胞破碎后离心收集包含体沉淀,菌体裂解液进行Ni-Chelsting亲和层析,Sephadex G-25凝胶过滤脱盐、去咪唑,DEAE Sepharose阴离子交换柱浓缩并去除碱性杂蛋白,Sephadex G-25凝胶过滤脱盐,肠激酶切割PACAP-PTRX融合蛋白。DEAE Sepharose阴离子交换层析除去残余酸性杂蛋白,成熟蛋白经Sephadex G-25分子筛和SPSepharose阳离子交换柱浓缩并除去杂蛋白,再冻干。
10、一种石斑鱼腺苷酸环化酶激活多肽重组蛋白,其特征是由权利要求8所述的重组工程菌株通过权利要求9所述的方法而获得的。
11、权利要求10所述的重组石斑鱼腺苷酸环化酶激活多肽在制备海水鱼类使用的生长与繁殖调控制剂中的应用。
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CN103145851A (zh) * | 2013-02-22 | 2013-06-12 | 暨南大学 | 重组蛋白PACAP38-NtA及其编码基因与应用 |
CN101506222B (zh) * | 2006-07-14 | 2013-10-02 | 健泰科生物技术公司 | 重组蛋白的重折叠 |
CN106132995A (zh) * | 2014-03-28 | 2016-11-16 | 诺维信公司 | 蛋白质晶体在低pH下的再增溶 |
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CN101506222B (zh) * | 2006-07-14 | 2013-10-02 | 健泰科生物技术公司 | 重组蛋白的重折叠 |
CN103145851A (zh) * | 2013-02-22 | 2013-06-12 | 暨南大学 | 重组蛋白PACAP38-NtA及其编码基因与应用 |
CN103145851B (zh) * | 2013-02-22 | 2014-07-02 | 暨南大学 | 重组蛋白PACAP38-NtA及其编码基因与应用 |
CN106132995A (zh) * | 2014-03-28 | 2016-11-16 | 诺维信公司 | 蛋白质晶体在低pH下的再增溶 |
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