Identification, Full-length CDNA Construction And Expression Of Part Coat Protein Gene Of Giardiavirus In Giardia Lamblia Isolated From Human In China

Giardiavirus were discovered firstly in Giardia lamblia isolated from human in China The characterization of the virus was identified. The full-length cDNA of giardiavirus was sequenced and constructed. The gene of pET-28c(+)- GLV1518-2322 was expressed in prokaryotic syste.Giardia lamblia were cultured in vitro Giardia lamblia BEIJ88/BTMR1/1 were successfully cultured in vitro. The body configuration were observed by light microscope and electron microscope. Growth state was very well in culture medium of TYI-S-33 and the vital force didn't decrease. Freezed in 10%DMSO, Giardia lamblia were cultured continuously and the growth propert didn't change. Giardia lamblia looked like tadpole and had two nuclei and four pairs flagellae. Giardia lamblia looked like demipear shape by transmission electron microscope. Its anterior was blunt round . Its posterior extremity was pegky gradually. Its rear was hemisphere shape. Its anterio abdomen depressed ieward to format chuck. There were many microvacuoles under body surface . Dactylogram structure was observed in vivo. Variable form Giardia lamblia was observed . Giardia lamblia cloned by binary fission. Giardia lamblia cyst was observed .It was oval-snap .The cyst wall was very thick .There was transparent air space between cyst wall and polypide .The characterization of the virus was identified Giardia lamblia virus was dsRNA virus,spherical shape and 36nm in diameter through identification. It lied not only in cytoplasm and nucleus but also in cyst. The virus particle lied in culture medium and around vacuoles. Virus coat filaments were also observed. Total nucleic acids from trophozoites of Giardia lamblia were extracted with phenol/chloroform for agarose gel electrophoresis and digested with DNase I and RNase A.According to the published nucleotide sequence of Giardiavirus.the pair of primers for RT-PCR were designed. After RT-PCR, The products were linked into pMD18-T vector, then cloned and sequenced. A particular extrachromosomal about 7.0kb band was observed after agarose gelelectrophoresis. The nucleic acid was not sensitive to DNase I, however, it was sensitive to RNase A (0.1 g/ml).And a fragment of 856bp was amplified by RT-PCR. The homology of Giardia lamblia BEIJ88/BTMR1/1 virus to that of sequences published by Wang (1993) in GeneBank was 98%. The activity of RNA dependent RNA polymerase was also detected in crude lysates of Giardiavirus.Full-length cDNA cloning was constructed According to the published nucleotide sequence of giardiavirus,six pair of primers for RT-PCR were designed. After RT-PCR, The products were linked into pMD18-T vector, then cloned and sequenced. Genome sequence of giardiavirus in Giardia lamblia isolated from human in China was 6273bp. The homology ofGiardia lamblia BEIJ88/BTMR1/1 virus to that of sequences published by Wang (1993) in GeneBank was 95% in the nucleotide,and 90% in the amino acid levels. Full-length cDNA cloning was developed by the six fragment linkaged at five intercut enzyme as Pvu I ,Xho I ,Kpn I ,Nhe I and Apa I .The gene of pET-28c(+> GLV1518-2322 was expressed in prokaryotic syste According to the sequence of Giardiavirus genome from China, the recombi- nant plasmid pET-28c(+)-GLVl 518-2322 coding part coat protein of Giardia- virus was developed. After it was transformed into E.coli strain DE3, then induced by IPTG ,32Da expression protein was found by SDS-PAGE. Expression product was 68.172% of total protein.After BalB/c Mice were immuned by the expression protein, antiserum of GLV1518-2322 was prepared.