ABSTRACT
The object of study was to clone the gene of ORFV ORF047 and study the eukaryotic expression and cell localization,making the theoretical basis for the subsequest screening of protein that interact with ORF047.ORF047 gene was amplificated by the specifical primer from the DNA of ORFV using PCR,the length was 735 bp,compared with L1 published in NC-005336.1,the homologies of the nucleotide acid sequence and amino acid sequence were 98.8％ and 98.8％.In order to defined the expression and location of the ORF047 gene in cell,the recombinant plasmid pEGFP-ORF047 was constructed and transfected into 293T cell,after 36 h,the green fluorescence could be observed under fluorescence microscope,and 54 kD protein was detected by western bloting.The plasmid of pHcRed1-Nuc,pHcRed1-Mito and pHcRed1-ER with the recombinant plasmid of pEGFP-ORF047 was cotransfected to veroE6 cell respectively,that fusion protein of ORF047 was mainly located in the cytoplasm,a small amount in the mitochondriabyconfocal microscopy analysis.
ABSTRACT
Lysozyme generally exists in animals,plants and microorganisms,and it is used as a natural anti?infection materi?al and one of the important non?specific immune factors in organisms. This paper reviews the progress of researches on its classifi?cation,gene structure and function,and expression regulation in Oncomelania hupensis,and on the factors affecting its activi?ties in recent years,in order to further discuss its distribution in O. hupensis.
ABSTRACT
<p><b>Objective</b>To construct eukaryotic expression plasmids of the Tac1 gene and explore the interaction between TAC1 and sperm-associated antigen 6 (SPAG6).</p><p><b>METHODS</b>RNA was extracted from the heart, liver, spleen, lung, kidney, brain, muscle, and testis of 10 Kunming male mice and, after reverse transcription into cDNA, the expression of Tac1 in the above tissues was observed by RT-PCR. Tac1/pEGFP-N2 and Tac1/pGADT7 recombinant plasmids were constructed and Tac1/pEGFP-N2 was transfected into CHO and COS-1 cells, followed by localization and detection of the protein expression of TAC1 by immunofluorescence staining and Western blot. The interaction between TAC1 and SPAG6 was determined by yeast two-hybrid experiment and Western blot.</p><p><b>RESULTS</b>Tac1 was expressed mainly in the testis, brain and heart. The results of restriction enzyme digestion and sequencing indicated successful construction of the recombinant plasmids, with the restriction fragment length of 390 bp. TAC1 was localized in the whole body of the CHO cells when transfected alone, but expressed in the microtubule of the cells when cotransfected with SPAG6, with the molecular weight of 40 000. Yeast two-hybrid experiment showed the colonies of TAC1 and SPAG6 on the culture plate without Leu, Trp and His, both contained in the yeast fusion protein.</p><p><b>CONCLUSIONS</b>The Tac1 recombinant plasmid was constructed successfully and the interaction between TAC1 and SPAG6 was confirmed with the plasmid.</p>