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Molecular Pathogens
MP2010, Vol.1, No.1
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(TaKaRa), 1 μL DNA, and then added ddH2O to 50μL.
Cycling conditions were performed as follows: Hot
start; initial denaturation at 95
℃
for 3 min, then
followed by 35 cycles each with denaturation at 94
℃
for 45s, anneal at 52
℃
for 30s , and elongation at
72
℃
for 60s, with a final elongation step of 72
℃
for
10min. Gel-purified amplicons were cloned into the
plasmid pGEM-T Easy vector (Promega) and
transformed into Escherichia coli JM109 competent
cells following the manufacturer's instructions.
Plasmids were extracted using the High Pure Plasmid
Isolation Kit (TaKaRa).
3.5 Sequence analysis
All the cloned transformants were sent to Shanghai
Sangon Biological Engineering Technology &
Services Co., Ltd. for sequencing. Then the sequences
were assembled and edited using the DNAMAN 5.2.9
Demo version software. Similarity searches were
performed
with
the
BLAST
program
(www.ncbi.nlm.nih.gov) using the default settings.
Predicted protein sequences were aligned using the
ClustalX 1.81 Program. A phylogenetic tree was
constructed by the neighbor-joining method
implemented in the Molecular Evolutionary Genetics
Analysis (MEGA) software with the Poisson
correction.
Acknowledgement
This research was supported by the National Public Welfare Scientific
Research Institution Foundation (SSCRI200714, SSCRI200804) and the
Hainan Provincial Natural Science Foundation (No.807039)
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