珍珠和贝壳形成机理的研究

Five cDNA fragments covering the complete genome of HCLV were obtained by RT-PCR, sequenced and inserted into pGEM-T vector, separately. A clone of the full-length cDNA of the complete genome of HCLV was obtained by subcloning all of the five cDNA fragments into pPoly2. The genome is 12,310 nucleotides in length and contains a large open reading frame encoding 3,898 amino acids.The sequence has been submitted to GenBank and its accession number is AF531433. Phylogenetic analysis based on genomic sequences and their deduced amine acid sequences of known ninteen CSFV strains showed that there is no relatonship between homology and virulence. SK6 cell was transfected with the gemone RNA of HCLV obtained by by transcription of the full-length cDNA with T7 polymerase and passaged four days later. A transfering plasmid of fluorescence-emitted recombinant HCLV was obtained by inserting the EGFP gene into the Npro gene of the cDNA of HCLV gemone in frame. SK6 cell was transfected with the RNA of the transfering plasmid and fluorescence-emitted SK6 cell was observed in the second passage..T7 RNA polymerase (T7pol) gene was amplified by PCR from the gemone of E.coli strain BL21(DE3) and cloned into the pGEM-T vector. A T7pol prokaryotic expression plasmid pET28T7 was constructed by subcloning the T7pol gene into vector pET28b(+). A eukaryotic expression of T7pol, pIRT7, was constructed by subcloning the T7pol gene into pIRES1neo vector. A cell line expressing the T7pol was obtained by trasfecting the pIRT7 into SK6 cell.　.　　The cDNA of porcine interleukin 6 (pIL-6) was amplified by RT-PCR from the total RNA extracted from the peripheral blood lymphocytes stimulated with Concavadin and cloned into the pGEM-T vector. Sequencing analysis showed that the encoding region of pIL-6 cDNA is 639pb including the stop coden. A prokaryotic expression plasmid of pIL-6, pETPIL6, was obtained by subcloning the encoding region of the pIL-6 mature peptide into pET-28a(+). The pIL-6 was expressed in pETPIL6-transformed BL21(DE3)LysS induced by IPTG with the yield accounting for 30.6～38.35% of the total bacterial protein..

1、用同位素研究占珍珠和贝壳成分95%的碳酸钙和4%的蛋白质合成、转运途径、分泌方式；2、研究泥色有机质珠与壳皮、骨珠与壳枝柱层、光珠与壳珍珠层形成机制；3、研究碳酸酢酶分布与活性；4、分离纯化该酶研究其动力学、功能基因；5、研究pH值、农药对功胞内外Ca^{+-}跨膜流动的影响。该研究对阐明珍珠和贝壳形成机理有重要理论意义，可为优质珠程峁├砺垡谰荨