刺激隐核虫小GTP酶Ran基因的克隆与表达

Cloning and expression of a Ran-like small GTPase gene from Cryptocaryon irritans

对刺激隐核虫(Cryptocaryon irritans)的小GTP酶Ran基因(Ci Ran)进行研究,以期为刺激隐核虫病原生物学研究及其防治提供理论基础。从刺激隐核虫滋养体/包囊前期cDNA文库中筛选出Ci Ran基因的克隆,利用生物信息学方法对CiRan基因及其编码的蛋白进行结构与功能的预测;通过逆转录PCR检测CiRan的mRNA。结果表明其在刺激隐核虫的各个发育阶段均有表达。对CiRan基因开放阅读框内的非通用密码子进行改造,并构建重组质粒p GEX-4T-1/CiRan,将其转化到大肠杆菌(E.coli)后成功表达分子量为51.3 kD的重组融合蛋白rCiRan。用rCiRan蛋白免疫鼠血清进行免疫印迹分析,结果表明,抗rCiRan血清能识别刺激隐核虫各期虫体的天然CiRan蛋白,其表观分子量为25.3 kD,与根据编码基因序列推测出的理论值相符;以rCiRan的抗血清做间接免疫荧光抗体实验,结果表明天然CiRan蛋白在幼虫的细胞质和细胞核内均有分布,且在核膜周围富集,佐证了该分子潜在的功能。

Ras-related nuclear proteins （Rans）, a family of small monomeric G proteins, are important in many cell activities, participating in the regulation of eukaryotic nucleocytoplasmic transport, DNA replication, mitotic spindle assembly, nuclear envelope dynamics, etc. In this study, a Ran gene （designated CiRan） was cloned from a cDNA library of Cryptocaryon irritans trophonts and characterized. The full-length CiRan cDNA was 771 bp, and contained an open reading frame （ORF） of 654 bp, encoding a putative polypeptide of 217 amino acids. The ORF contained 6 TAAs and 2 TAGs, which encode glutamine in the ciliate rather than stop codons, as in the universal genetic code. The calculated molecular weight of the CiRan protein was 25.3 kD, with a pI of 8.9. The sequence information has been deposited in GenBank under accession number KP662712. A bioinformatic analysis predicted that the deduced CiRan protein con- tains a typical Ran domain, extending from residue 11 to residue 174. A GTP/Mg2＋-binding site occurs at residues 17-153, a Switch I area at residues 30-50, a Switch II area at residues 67-87, a G1 ring at residues 17-26, a G2 ring at residues 40-42, a G3 ring at residues 65-70, a G4 ring at residues 120-125, and a G5 ring at residues 148-152. The predicted structural characteristics confirm that CiRan is a member of the Ran protein family. A phylogenetic tree of CiRan and 10 Ran proteins from other species revealed that CiRan is closely related to the Ran proteins of Paramecium tetraurelia, lchthyophthirius multifiliis, and Tetrahymena thermophila. Reverse transcription-PCR showed that the Ci- Ran gene is expressed in all the developmental stages of the C. irritans life cycle. After the non-universal codons, TAA and TAG, in the ORF of the CiRan cDNA were modified to CAA and CAG, respectively, and the ORF DNA was amplified and inserted into the EcoR I-Xho I restriction sites of the bacterial expression vector pGEX-4T-1, generating the recombinant plasmid pGEX-4T-1/CiRan, which was used to transform Escherichia coli. The transformed E. coli was induced with isopropyl-13-D-1-thiogalactopyranoside to express the recombinant protein rCiRan, as a fusion protein with gluthathione S-transferase （GST）. After purification with glutathione Sepharose 4B, rCiRan was used to intraperitoneally immunize specific-pathogen-free Kunming mice to prepare a polyclonal antibody against rCiRan. Another group of mice was immunized with GST to prepare a polyclonal antibody against GST for the negative control. The results of a western blotting analysis showed that the polyclonal antibody against rCiRan recognized the native CiRan protein in lysates of different developmental stages of C. irritans. The molecular mass of native CiRan was 25.3 kD, which is consistent with the value calculated from the coding sequence. The native CiRan protein in C. irritans theronts was localized with an indirect immunofluorescent antibody assay with the antibody directed against rCiRan, showing that native CiRan was not only distributed in the cytoplasm of C. irritans theronts, but also in the nuclei. It was especially abundant around the nuclear membrane, implying that the protein has nu- clear-membrane-related functions. This study extends our theoretical understanding of the biology of the pathogen C. irritans, and lays the foundation for future studies of the functions of CiRan, which may be important in the prevention of C. irritans infection and the control of cryptocaryonosis.