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Cloning, Expression and Activity Analysis of Recombinant Triosephosphate Isomerase from Haemonchus Contortus & Recombinant Glutamate Dehydrogenase Activity Analysis

Author: SuHuiMin
Tutor: LiXiangRui
School: Nanjing Agricultural College
Course: Preventive Veterinary Medicine
Keywords: Haemonchus contortus Triosephosphate isomerase Glutamate dehydrogenase Gene cloning Enzyme activity
CLC: S852.7
Type: Master's thesis
Year: 2010
Downloads: 7
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Abstract


Haemonchus contortus belongs to the nematoda phylum, strongylida order, trichostrongylidae family, and Haemonchus genus. It is a blood-feeding nematode parasite that infects the abomasums of ruminants. Infection can lead to anaemia and the death of the host, especially lambs, in severe cases, which cause significant economic loss of husbandry industry. Haemonchosis’s control is, so far, carried out by combining anthelmintics with grazing management. However, excessive and uncontrolled uses of chemical drugs have resulted in the emergence of anthelmintics-resistant strains of the parasite. So, it is quite necessary to develop alternative strategies for the control of H.contortus parasite. Vaccination seems to be the ultimate, effective and sustainable strategy to controlling this parasite.Triosephosphate isomerase (TPI), a kind of dimeric enzyme, has an important role in glycolysis, gluconeogenesis, fatty acid synthesis, and the pentose shunt, converts glyceraldehyde-3-phosphate to dehydroxyacetone phosphate. This thesis describes the cloning and sequence analysis of the full length HcTPI cDNA gene, prokaryotic expression and characterization of the recombinant HcTPI protein.1. Cloning and sequences analysis of Haemonchus contortus TPI cDNA geneGene specific primers were designed based on H.contortus expressed sequence tag (EST) of TPI in GenBank (Accession numbers:CB015183). The 3’end and 5’end cDNA products were generated by 3’and 5’-RACE (rapid amplification of cDNA ends) procedure, respectively. The full length sequence of HcTPI gene was obtained by joining overlap sequences of 5’ends, EST and 3’ends with DNAStar programmer. The 986bp long HcTPI cDNA gene had an open reading frame of 744bp, 5’untanslated region (UTR) of 64 bp and 3’UTR of 178bp. According to this ORF, the specific primers were designed and the 744bp products was amplified by reverse transcriptase- polymerase chain reaction (RT-PCR) with total RNA extracted from the adult worms of H.contortus as template. Comparisons and analyses of the nucleotide acid sequences indicated that the amplified gene encodes a 247 amino acid protein with an isoelectric point (pI) of 8.18 and predicted theoretical molecular mass of 27.47 kilodalton. The protein could easily be aligned with members of TPI superfamily.2. Expression of HcTPI cDNA in E. coli and characteristic of the recombinant proteinBy the means of DNA recombination technique, HcTPI gene was cloned into prokaryotic expression vector pET32a (+) and then transformed into Rosetta (DE3). The recombinant protein was expressed by inducing with IPTG. As the results of SDS-PAGE indicated, the recombinant protein was successfully expressed in E. coli with the relative molecular weight of about 45kDa and existed in both of the supernatant and inclusion body. Western blot analysis showed that the product was recognized by goat serum infected with H.contortus. The enzyme activity was assayed in the glyceraldehyde 3-phosphate (GAP) to dihydroxyacetone phosphate(DHAP) direction in a coupled assay performed with a-glycerophosphate dehydrogenase (a-GPDH), the result showed the optimum reaction temperature and pH of recombinant TPI was 40℃, PH 7.5, respectively. The measurement of enzyme kinetics revealed that the Km was 0.96mmol/L and the Vmax was 0.341 mmol/(L-min).3. Expression of HcGDH in E. coli and characteristic of the recombinant proteinpET-28a(+)-HcGDH plasmid was transformed into BL21(DE3), and then induced by IPTG. SDS-PAGE gel showed that a relative 63kDa recombinant protein was expressed in the form of inclusion bodies. Preparative protein was purified with purification kit and refolded utilizing urea step by step as solvent. Analysis of glutamate dehydrogenase activity of the recombinant protein indicated that its activity was attributed to NAD-dependent GDH, and the optimum reaction temperature was 40℃and pH was 8.0. Enzyme kinetics result showed that the Km was 1.26mmol/L and the Vmax was 0.055 mmol/(L·min).

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Livestock parasitology
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