Basic HTML Version
Molecular Pathogens (online), 2011, Vol.2
ISSN 1925-1998 http://mp.sophiapublisher.com
38
(TIANGEN, China). The purified DNA fragments
were ligated into the PMD19-T vector (TaKaRa
Biotechnology, China) following the manufacturer’s
instruction, and used to transform
E
.
coli
DH5α
.
The
positive clones were confirmed by PCR and restriction
enzyme digestion and named as Y/PMD-T before
sequencing. Two clones from independent PCR
reactions were sequenced from both directions.
The primers PS-E and PA-E were designed according
to previous studies. We used these primers and
template Y/PMD-T for PCR amplification. The
purified DNA fragments were ligated into the
PMD19-T vector and transformed into
E
.
coli
DH5α.
The positive clones were confirmed by PCR and
restriction enzyme digestion and named as
CP-Y/PMD-T.
3.6 Sequence analyses
Sequence homology analysis was conducted with
DNAMAN; Phylogenetic trees were constructed by
neighbor-joining (NJ), minimum evolution (ME), and
maximum parsimony (MP) method in the MEGA
(version 4.1) software (Mushegian and Koonin, 1993).
Bootstrap analyses with 1000 replicates were
performed to evaluate the significance of the interior
branches.
3.7 Construction of prokaryotic expression vectors
of ASPV CP gene
The recombinant plasmid CP-Y/PMD-T was cloned into
the
Nco
I and
Sal
I restriction sites of the expression
vector pET-28a (+). The insert and vector were ligated
with T4 DNA ligase and then was used to transform
E
.
coli
DH5α. The correct recombinant positive clone was
selected and sequencing to verify the correctness of ORF
and designated ASPV-CP-Y/PET.
3.8 Induction expression and SDS-PAGE electroph-
oresis analysis of ASPV CP gene
The recombinant plasmid ASPV-CP-Y/PET-28a was
transformed into
E
.
coli
BL21 (DE3), and harboring
ASPV-CP-Y/PET was grown in 3 mL LB liquid
medium supplemented with kanamycin (50 g/mL), 37
℃
overnight and then 1: 100 diluted, vaccination into
fresh 10 mL LB liquid medium with the
corresponding antibiotic with shaking at 37
℃
an
OD
600
of 0.6~1.0. The culture was induced with IPTG
(isopropyl-β-D-thio-galactoside) and the final concen-
tration of 1 mM. The cells were grown for an
additional 4~6 h to allow expression of the recombinant
protein and then harvested by centrifugation at 8000
r/min for 5 min and resuspended in TE solution (pH
8.0), adding volume of 2
×
SDS buffer, and then the
cells were boiled for 3 min, centrifuged at 12 000 r/min
for 10 min. The recombinant protein was detected by
SDS-PAGE using 12% polyacrylamide gel. Uninduced
recombinant clone and
E
.
coli
BL21 (DE3) host cells
(with and without IPTG) were used as controls. Briefly,
the gel was stained with Coomassie brilliant blue R-250
for 1 h and destained in acetic acid until a clear
background was seen.
Author’ Contributions
JXN was responsible for experimental design and the
experiment direction; NL and JXN were responsible for data
analysis, paper writing and modification. Both the authors had
read the final version of this paper and agreed with the authors’
credits.
Acknowledgements
This study was supported by National Natural Science Foundation of
China (30360066), the National Key Technologies R&D Program of
China (2003BA546C), the Foundation Science and Technology
Commission Xinjiang Production and Construction Crops, China
(NKB02SDXNK01 SW) and Natural Science and Technology
Innovation of Shihezi University, China (ZRKX200707).
References
Deng X.Y., and Wang G.P., 2002, Advances in research on pear viruses,
Journal of Fruit Science, 19(5): 321-325
He M., Li H.S., Zhao Y., Niu J.X., and Ma B.G., 2004, A method of rapidly
extracting total RNA from Korla pear, Shihezi, Journal of Shihezi
University, 22(6): 474-476
Hou Y.X., 2005, Research on fast detection of
Apple stem pitting virus
in
Pyrus pyrifolia
and clone and the prokaryotic expression of its coat
protein gene, Thesis for M.S., Huazhong Agricultural University,
Supervisors: Wang G.P., and Hong N., pp.29-43
Jelkmann W., 1994, Nucleotide sequences of
apple stem pitting virus
and of
the coat protein gene of a similar virus from pear associated with vein
yellows disease and their relationship with potex- and carlaviruses, J.
Gen. Virol., 75(7): 1535-1542
http://dx.doi.org/10.1099/0022-1317-75-7-1535 PMid:8021584
Jelkmann W., Kunze L., Vetten H.J., and Lesemann D.E., 1992, cDNA
cloning of dsRNA associated with apple stem pitting disease and
evidence for the relationship of the virus-like agents associated with
apple stem pitting and pear vein yellows, Acta Horticulture, 309: 55-62
Leone G., Lindner J.L., van der Meer F.A., Schoen C.D., 1998, Sympotoms
on apple and pear indicators after back-transmission from
Nicotiana
occidentalis
confirm the identity of
apple stem pitting virus
with pear
vein yellows virus, Acta Hortic., 472: 61-65
Li L.L., Dong Y.F., Zhang Z.P., Zhang Z.H., Fan X.D., and Pei G.Q., 2010a,
RT-PCR detection and molecular variability of
apple stem pitting virus
,
Acta Horticulturae Sinica, 37(1): 9-16
Li L.L., Yang H.Y., Dong Y.F., and Gao Y., 2010b, Production of antiserum