草鱼干扰素基因克隆及其在衣藻中表达的研究

In this project, the fellowing researches had been performed: (1) Type I interferon of grass carp (Ctenopharyngodon idellus ) was purified by means of gel chromatography, anion exchange chromatography, high performance liquid chromatography and gradient polyacrylamide gel electrophoresis. The terminal amino acid sequence of the interferon was analysed and the degenerate primer of the gene was synthesized. (2) the cDNA library of grass carp leucocytes was constructed. For this, total RNA was extracted from the head-kidney leukocytes using single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. mRNA was isolated with paramagentic particles method. The cDNAs were synthysized after a series of enzyme-catalyzed reaction, and then ligated with EcoRI adaptors. Over 500bp fragments were collected and cloned intoλgt10 andλZAPII vector after removing short cDNA segments and excess adaptors through Sephacryl S-400 resin and spin columns. After package of ligated DNA in vitro, the constructed cDNA library contained 2.1×107 recombinants, in which more than 92% clones were recombinant. The insert size ranged from 0.5 to 3 kb. It demostrated that the cDNA library is of high quality and can be used a valuable resource for studying immune gene expression and screening genes associated with the immune response of grass carp.(3) cDNA coding grass carp interferon was cloned and sequenced. The full length of grass carp interferon cDNA was 492bp coding for 164 amino acids, the deduced molecular weight of the protein was about 20kd, several potential N-glycosylation sites were observed in the deduced peptide sequence which suggested that grass carp interferon was a glycoprotein. Analysed by blastn software, no significant homology of nucleotide and amino acid sequence between the interferon of grass carp and human or other higher vertebrates were found, indicating the specificity of fish interferon in phylogency. (4) A Chlamylomonas reinhardtti chloroplast transformation and expression vector was constructed. The vector contains homologous fragments, clpP-trnL-petB, at the side of gene chlL for directinal integration of foreign genes into the genome of chloroplast, a multiple cloning sites between chlL5' and chlL3' untranslated regulatory for insertion of foreign target genes and a aadA gene cassette designed to express spectinomycin resistance for recombinants screen. The grass carp interferon gene was recombinated into the vector, transformed into the wild type Chlamylomonas reinhardtti cells and expressed in Chlamylomonas reinhardtti chloroplast under the control of the promotor of 5'atpA and terminator of 3'rbcL. (5) The immunomodulation function of grass carp interferon was investigated, the results showed that grass carp interferon was an important immunomodifier which was able to increase the activity of macrophages and lymphocytes in fish. (6) A new type II interferon (γinterferon) and a new natural immunoreactive agglutinin was discovered in grass carp in the process of isolation for type I interferon. The results of the project were important for understanding the origin and phylogency of interferon, and application of fish interferon in the future.

本项目拟在申请者已分离纯化出草鱼干扰素等工作的基础上，进一步构建草鱼白细胞cDNA文库，开展干扰素基因克隆及序列分析，同时构建衣藻叶绿素遗传转化载体，将草鱼干扰素基因定向导入衣藻叶绿素基因组表达，利用转基因衣藻作为生物反应器，生产鱼类干扰素，应用于鱼类防治研究，以丰富鱼类基因工程内容，为鱼类病害防治开辟新的途经。