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Cloning and Expression Analysis of Hsp70 Gene from Freshwater Planarian Dugesia Japonica

Author: LouZuo
Tutor: ChenGuangWen
School: Henan Normal
Course: Zoology
Keywords: Planarian Heat shock protein 70 Clone Protein expression Polyclonal antibody preparation
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 46
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Abstract


Heat shock protein 70 (Heat Shock Protein 70, HSP70) is an important member of the heat shock protein family. Numerous studies have confirmed: an important role in the HSP70 protein folding, membrane transport, and degradation of misfolded proteins, such as cell proliferation, differentiation, and nervous system is closely related to, and its expression by environmental stresses and physiological stress impact. Important as the body's protective protein, HSP70 has not been reported in the planarian. In this study, the RACE technique cloned for the first time in Japan Dugesia the hsp70 (Djhsp70) full-length cDNA sequences registered in GenBank accession number EU380241. Genomic DNA and cDNA as template to amplify the open reading frame (ORF), the same sequencing results described Djhsp70 no introns. 2066bp, Djhsp70 cDNA full-length open reading frame (ORF) 1947bp, encoding amino acids 648 molecular weight 71.18kDa. BLAST search analysis of currently known found Djhsp70 nucleotide sequence of HSP70 family members containing eukaryotic HSP70 family proteins the three label the sequence (IDLGTTYS, DLGGGTFD, IVLVGG) as well as the end of highly conserved sequence EEVD of highly homologous to the amino acid sequence. This is in line with the reported HSP70 characteristics. Build HSP70 phylogenetic tree based on the amino acid sequence of the HSP70 reported results show that the planarian Mollusca mussels recently, near invertebrates, worms and flies far away. The above results planarian new taxonomic status - crown wheel animal is consistent. We research Stress of Djhsp70 expression using semi-quantitative RT-PCR technology. The results showed that: one week after feeding, 18 ℃ culture planarian normal control. Heat treated condition, 10 ℃ expression were higher than the control group, a slight increase when the temperature is raised to 30 ° C continuous 24h, its expression was significantly increased compared with the control group, a significant difference (P <0.05); 32 ° C continuous 48h expression amounted to nearly twice that of the control group. Under conditions of starvation, hunger 15d expression compared with the control group had no significant change, hunger 30d expression was significantly increased after feeding 60d expression rapidly decreased. Cutting processing conditions, cutting 12h express higher than the control group, a significant increase (P <0.05), and the largest decrease, 48h 36h expression. Thus, we think, Djhsp70 inducible hsp70 gene, with the planarian hot patience, closely related to anti-hunger as well as tissue repair. For in further research DjHSP70 positioning on histological, this experiment was constructed Djhsp70 C-terminal peptide of prokaryotic expression vector, about 21kDa fusion protein expressed under IPTG induction. Ni-Agarose resin fusion protein was purified, electrophoretic display purified fusion protein of high purity, more than 95% by the grayscale scan analytical purity. Purified fusion protein antigen to immunize New Zealand white rabbits, the successful preparation of rabbit anti-HSP70 polyclonal antiserum. Western blotting analysis shows that the antiserum with planarian HSP70-specific binding, no cross-reactivity with other heat shock proteins, indicating that the specificity of the antibody. Dlot Blot assay antibody titer of 1:500. The antiserum further testing at the protein level thermal stimulation, hunger-induced, as well as cutting injury HSP70 expression in planarians, the results are consistent with the RT-PCR test results. In summary, this study successfully clone Djhsp70 full-length cDNA sequences confirmed DjHSP70 is an inducible heat shock protein, no introns. MRNA and protein expression levels proved the HSP70 planarian hot patience stress phenomenon is closely related to the anti-hunger and tissue repair. Polyclonal antibody prepared successfully laid a good foundation for the further study of the positioning of HSP70 in planarian tissue and other biological functions.

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