Identification Of Rhesus Macaque Entamoeba Histolytica By PCR And Gal/GalNac Lectin Hgl LC3 Gene Clone, Prokaryotic Expression

An Entamoeba sp.strain was isolated from an adult female rhesus macaque with symptom including dysentery and diarrhea.Through observing the morphous and measuring the sizes of amoeba trophozoites and cysts,we diagnosed the isolated strain as E.histolytica/E,dispar.Using the species-specific primers for E.histolytica and E.dispar (p11 plus p12 and p13 plus p14,respectively)for PCR diagnosis,we diagnosed this case as E.histolytica natural infecting in rhesus macaque.Base on the published nucleotide sequence of human E.histolytica hgl gene,a pair of PCR primers was designed and synthesized.Total genome DNA isolated from the Entamoeba sp.strain was used as template to PCR amplification.The PCR production was cloned into PMD18-T vector,and 1761bp DNA fragment was obtained by sequencing.By blasting the homologous sequences in Genbank databases,DNA sequence was confirmed as the E.histolytica hgl gene sequence.The determined nucleotide sequences were compared pair wise for similarity,the results showed that the sequence of hgl gene from rhesus macaque E.histolytica was between 93.44%-96.21%percent identify to the human E.histolytica hgl genes,was 87.44%percent identify to the human E.Dispar hgl gene.This DNA sequence encodes a protein consisting of 587 amino acid(AA).The molecular mass of polypeptide was 65.514KD,its isoelectric point was 5.17,also has the strong hydrophilicity and antigenicity.The sequence produced in this study was deposited in Genbank(Accession No.EU244479).By the technology of DNA recombination,the LC3 segment of hgl gene was cloned into expression plasmid pET-32b(+),and was transformed to E.coli BL21.The recombinant bacteria were induced to expression through the changes of IPTG.The results showed that a new protein band was found in SDS-PAGE with molecular mass of about 66KDa,which was consisted of a 46KDa protein of the hgl gene and pET-32b(+)(20KDa). The amount of proteinum was to peak at 3h after IPTG induced.There were almost no difference to amount of proteinum in different IPTG concentration,all be high.