扇贝丝氨酸蛋白酶抑制剂基因CfKZSPI及Aikunitz的克隆与重组表达

IOCAS-IR > 海洋环流与波动重点实验室

	扇贝丝氨酸蛋白酶抑制剂基因CfKZSPI及Aikunitz的克隆与重组表达
其他题名	Molecular cloning, expression of serine protein inhibitor genes CfKZSPI and Aikunitz from scallops, and inhibitory activity of the recombinant proteins
	王波
学位类型	博士
	2008-06-10
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	栉孔扇贝海湾扇贝丝氨酸蛋白酶抑制剂 Kazal Kunitz Cdna克隆重组表达抑制蛋白酶活性
摘要	扇贝养殖是我国重要的海水养殖产业，然而自1997 年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且严重影响了该产业的健康发展。丝氨酸蛋白酶抑制因子及丝氨酸蛋白酶在无脊椎动物的免疫应答中起着核心作用，它们的协同作用直接导致外界病源入侵的信号转导和级联放大，并进一步激活一系列防御体系，如黑化反应、血液凝结和抗菌肽的合成等。因此，克隆扇贝参与免疫防御的丝氨酸蛋白酶抑制剂基因并对其功能进行研究，将有助于进一步研究扇贝的免疫防御机制，丰富和发展无脊椎动物免疫学的内容。运用大规模EST技术和RACE技术从栉孔扇贝中克隆出一个Kazal型丝氨酸蛋白酶抑制剂基因，定名为CfKZSPI。该基因cDNA序列全长1788bp，其中5' 非编码区（Untranslated Region, UTR）为97 bp，3' UTR161 bp，有一个典型的多聚腺苷酸信号序列（AATAAA）和一个ploy A 尾巴，开放阅读框（Open Reading Frame, ORF）含有1530 bp，编码509 个氨基酸残基。对其推测氨基酸序列进行分析，发现其中包括22个氨基酸残基组成的信号肽序列和12个Kazal型丝氨酸蛋白酶抑制剂结构域。采用QRT-PCR（quantitative real time PCR）对鳗弧菌浸泡刺激后栉孔扇贝血淋巴中CfKZSPI 的 mRNA表达量进行了检测，发现其mRNA 的表达量在鳗弧菌刺激后3h明显上升，达到空白组的43.6倍；然后在6h时有所下降，为空白组的15.0倍；随着菌刺激时间的增长，CfKZSPI基因的 mRNA 表达量急剧增加，在刺激后8h，12h，24h分别达到空白组的174.1，207.8，675.4倍。统计分析发现3h（P=0.019<0.05）和12h（P=0.020<0.05）时，CfKZSPI基因mRNA表达量与空白组差异均显著。为了研究栉孔扇贝CfKZSPI的蛋白活性，将其第十二个结构域克隆到pET-32a（+）载体中，转化大肠杆菌Rosetta-gami（DE3）表达菌株，获得可溶性表达的蛋白rCfKZSPI-12，对其进行抑制蛋白酶活性的分析，发现其对胰蛋白酶有很强的抑制活性，而对凝血酶没有抑制活性。当rCfKZSPI-12与胰蛋白酶分子比率为1：1时，约90%的蛋白酶活性被抑制。运用狄更斯作图法研究rCfKZSPI-12对胰蛋白酶的抑制能力，结果发现其对胰蛋白酶的抑制常数为173 nmol L-1。采用同样方法从海湾扇贝cDNA文库中克隆出一个Kunitz型丝氨酸蛋白酶抑制剂基因，定名为Aikunitz。该基因全长632 bp，其中5' UTR 为105 bp，3' UTR 为 245 bp，有一个典型的多聚腺苷酸信号序列（AATAAA）和一个ploy A 尾巴，ORF 含有282 bp，编码93 个氨基酸残基。推测的氨基酸序列N末端有一个20个氨基酸残基组成的信号肽序列，成熟蛋白包括一个Kunitz型丝氨酸蛋白酶抑制剂结构域。采用QRT-PCR对鳗弧菌和藤黄微球菌感染后海湾扇贝血淋巴中Aikunitz 的mRNA的表达量进行了检测，结果发现其在鳗弧菌刺激后3h到9h持续上升，9h时表达量为PBS对照组的4.49倍（P=0.008<0.05），然后开始下降，在72h时表达量为对照组的0.24倍（P=0.021<0.05）；而在藤黄微球菌刺激后3h到12h其表达量上升，其中6h时为空白组的5.95倍（P=0.0004<0.01）；12h以后迅速下降，其中24h的表达量为对照组的0.38倍（P=0.028<0.05）。将Aikunitz基因编码的成熟蛋白按照重组CfKZSPI-12的方法进行重组表达，并对重组蛋白进行抑制蛋白酶和抑菌活性分析。结果发现其对胰蛋白酶和弹性蛋白酶两种丝氨酸蛋白酶都没有抑制作用。抑菌实验同样发现，重组Aikunitz 对供试的革兰氏阳性菌藤黄微球菌和革兰氏阴性菌鳗弧菌和大肠杆菌都不显示明显抑菌活性。
其他摘要	Scallop aquaculture is a big industry and contributes enormously to the economic development of coastal provinces in China. Since the summer of 1997, large-scale mortality of cultured scallop has caused catastrophic losses to scallop aquaculture, which resulted in the drastic decrease of the production. Serine proteases inhibitors (SPI) together with serine proteases play a central role in the invertebrate immune response. They are mainly involved in signaling and amplification cascades that lead to the activation of some defense processes, such as melanization, coagulation and induction of antimicrobial peptides. Therefore, the cloning, expression and functional study of the two SPI genes from scallop will help us to understand the scallop’s immune defense mechanisms, and provide new information for the development of marine invertebrate immunology. A novel Kazal-type SPI gene was cloned from Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 bp, consisted of a 5’untranslated region (UTR) of 97 bp, a 3’ UTR of 161 bp including canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1530 bp encoding a polypeptide of 509 amino acids. The deduced amino acid sequence of CfKZSPI contained a putative signal peptide of 22 amino acids and twelve tandem Kazal domains. The temporal expression of CfKZSPI in haemocytes after Listonella anguillarum challenge was recorded by quantitative real time PCR (QRT-PCR). The expression of CfKZSPI was up-regulated to 43.6-fold against the blank at 3 h post-challenge, decreased to 15.1-fold at 6 h, and then increased again and reached 174.1-fold, 207.8-fold and 675.4-fold at 8 h, 12 h, 24 h, respectively. The statistical analysis revealed that the expression level of CfKZSPI at 3 h and 12 h was significantly higher than that in the blank group. These results indicated that CfKZSPI was involved in the immune response in Zhikong scallop. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCfKZSPI-12) displayed significant inhibitory activity against trypsin, while no activity against thrombin. Through the method of Dixon plot the inhibition constant (Ki) of rCfKZSPI-12 to trypsin was obtained to be 173 nmol L-1. A Kuintz-type SPI (Aikunitz) was cloned from the cDNA library of Bay scallop Argopecten irradians with the same method. The full-length cDNA of Aikunitz gene was of 632 bp, consisted of a 5’ UTR of 105 bp, a 3’ UTR of 245 bp and an ORF of 282 bp encoding a polypeptide of 93 amino acids. The deduced amino acid sequence was consisted of a signal peptide of 20 amino acids and a Kunitz domain. The temporal expression profiles of Aikunitz in haemocytes after Listonella anguillarum and Micrococcus luteus challenge were recorded by QRT-PCR. The mRNA level after Listonella anguillarum challenge was up-regulated from 3h to 9h, and reached 4.49-fold (P=0.008<0.05) against the control at 9h, then down-regulated, and reached 0.24-fold (P=0.021<0.05) against the control at 72h. The mRNA level after Listonella anguillarum challenge was up-regulated from 3h to 12h, and reached 5.95-fold (P=0.0004<0.01) against the control at 6h, then down-regulated rapidly after 12h, and reached 0.38-fold against the control at 24h. Aikunitz was recombined and expressed to investigate its inhibitory activity. The purified recombinant protein (rAikunitz) did not display inhibitory activity against the two potential serine proteinases, trypsin and elastase, nor to the growth of Gram-positive bacteria Micrococcus luteus, or Gram-negative bacteria Listonella anguillarum and E.coli.
页数	107
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1357
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	王波. 扇贝丝氨酸蛋白酶抑制剂基因CfKZSPI及Aikunitz的克隆与重组表达[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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