ABSTRACT
On the basis of the sequencing of the N-terminal amino acid of the crystal protein, a nucleotide acid fragment harboring a novel nematicidal gene cry6Aa2 was obtained from Bacillus thuringiensis strain YBT-1518. This fragment contains two ORFs: orf1 and orf2, while a "stem-loop" exists between orf1 and orf2. BLAST showed the similarity of orf1 nucleotide acid sequence with cry6Aa1 is 98%, and has been deposited in the Genbank database (Acc. No. AF499736). The cloning fragment was transferred to crystal negative mutation strain BMB171 by E. coli-Bt shuttle vector pHT304. A 54kDa protein with similarity to strain YBT-1518 was detected in recombinant strain, and rice shaped crystal was detected with transmission electron microscope. Bioassay indicated the LC50 of recombinant strain against second lavae juvenile of Meloidgyne hapla is 9.47 microg/mL, nearly equal to the original strain YBT-1518 (10.74 microg/mL).
Subject(s)
Anthelmintics/pharmacology , Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Endotoxins/genetics , Hemolysin Proteins/genetics , Recombinant Proteins/biosynthesis , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/pharmacology , Base Sequence , Cloning, Molecular , Endotoxins/pharmacology , Escherichia coli/genetics , Hemolysin Proteins/pharmacology , Molecular Sequence Data , Recombinant Proteins/pharmacology , Tylenchoidea/drug effectsABSTRACT
OBJECTIVE: To explore a new method for determining hepatitis B virus (HBV) genotypes B-D with real-time fluorescence-based PCR. METHODS: The PCR primers and probes were designed according to the analysis of 143 complete HBV genomes from GenBank and on the basis of a search for genotype-specific nucleotide sequences which were conserved in the 3 HBV genotypes. Real-time fluorescence-based PCR was performed for HBV genotyping of 128 samples collected from Lanzhou. Three samples of each genotype of B, C and D were randomly selected and their S gene was sequenced for confirmation of the results of PCR-based method. RESULTS: Real-time PCR identified genotype B in 26 (20.3%), genotype C in 92 (71.9%), and genotype D in 10 (7.8%) cases. The sequencing results of the S gene of 18 PCR-produced clones were in complete consistence with those of fluorescence-based PCR. CONCLUSION: The real-time PCR method is convenient, highly sensitive, rapid and accurate, especially suitable in clinical and large-scale epidemiological studies.
