Eukaryotic Expression Of The Cysticercus Cellulosae Heat Shock Protein And Product Analysis

As the larvae of taenia solium, cysticercus cellulosae, always causes cysticercosis when parasitize in human being, and this disease can also be named as Cysticercosis cellulosae. Cysticercus cellulosae can always invades every organ of human being, may survive in human body up to20years when it becomes adult, and this causes great threat to human health. At present, few basic research about cysticercus cellulosae, let alone some reports about the mechanism of this disease. Heat Shock Protein(HSP) are widespread exist in the original nuclear and eukaryotic cells, it can always generates a kind of highly conservative peptide protein molecule which with important physiological function. HSP can not only partcipate in the intracellular material transportation, the correct folding, degradation and repair of protein, but also relates with evade from the host immune system and enhance the toxicity of parasite. HSP gradually become a new target spot of the diagnosis and treatment about parasite disease. In order to deeply research HSP at the molecular level, to confirm the relationship between HSP and host immune reaction caused by taenia solium, we successfully constructed the eukaryotic restructuring plasmid of pVAXI-HSP35.6, and track its expression situation in mammalian cells by using enhanced green fluorescent protein. Preliminarily demonstrated that the taenia solium HSP can express product in eukaryocyte, and the product with highly activity. Research in detail as follow:1. The construction of Taenia Solium HSP35.6eukaryotic expression restructuring plasmidAccording to the sequence of Taenia Solium HSP35.6mRNA in the NCBI, we design the specific amplification primer with the help of Primer Premier5.0and DNAman. We conduct double enzyme digestion to the product of PCR and eukaryotic expression vector pVAXI, switches to competent Escherichia coli DH5a after combined. Identify and enzyme digestion the positive restructuring plasmid pVAXI-HSP35.6. Design another primer with the same method, to get enhanced green fluorescent protein(EGFP) by PCR, after double enzyme digestion, we combined it with pVAXI-HSP35.6, and got the positive restructuring pVAXI-HSP35.6-EGFP. After enzyme digestion appraisal to the restructuring plasmid, we submit it to sagan corporation to test the validity of purpose sequences. The homeology is100%between the augmented sequence after identified by sequencing appraisal and nucleotide sequence reported in Genebank. It means that the eukaryon restructuring plasmid of Taenia Solium HSP35.6has been successfully established.2. Eukaryotic expression of the Taenia Solium HSP35.6gene.The selected positive recombinant plasmid was transfected into Vero cell by LipofectamineTM2000, with a blank control of Vero cell without transfection. After48hours, green fluorescent could be seen in vero cells under the fluorescence microscope, but not in negative and blank. It means that the green fluorescent protein expressed successfully. In order to test the heat shock protein whether expressed or not, analysising the expression product activity was followed by.3. Analysis about the product of Taenia Solium HSP35.6eukaryotic expression.Collecting the cell successfully transfected(positive), and then conducted with RIPA lysis solution and test by SDS-PAGE and Western-blot. There is a band sizes35.6KDa in Western-blot after the SDS-PAGE cataphoresis, and this demonstrated that it can be identified by the swine positive serum of infected cysticercus cellulosae. The conclusion shows that the HSP and green fluorescent protein efficiently fuse-expressed, and the specificity of Taenia Solium HSP expression product is definitely high. This experiment has successfully constructed the eukaryon restructuring plasmid of Taenia Solium HSP, making the protein express efficiently in eukaryon, providing reliable evidence for futher research about the key of Taenia Solium invading the host.