Basic HTML Version
Molecular Pathogens (online), 2011, Vol.2
ISSN 1925-1998 http://mp.sophiapublisher.com
37
Recently, the antiserum for effective detection of
ASPV had not been produced. Hou et al (2005) fast
detection of ASPV
in
Pyrus pyrifolia
and clone and
the prokaryotic expression of its coat protein gene. Li
et al (2010b; 2010 c) found that the complete CP gene
of ASPV was expressed in
Escherichia coli
using gene
recombinant method, and the antiserum was produced
after the rabbit was immunized with purified protein
and production of antiserum of ASPV based on
antigenic epitopes technique, but only partial positive
samples could be detected with the antiserum.
Nucleotide sequence of different isolates of ASPV CP
genes have a high variation, and lead to existence
difference serological of ASPV, and thus influence the
detection results. We generally believed that amino
acid sequence variations of ASPV CP gene may cause
epitope variants, and then were divided into different
ASPV serotypes. Therefore, in order to improve the
accuracy of ASPV detection, it is necessary to
preparation of different ASPV antiserum. In this study,
we have successfully constructed ASPV CP gene into
prokaryotic expression vector PET-28a, and specific
expression of a 42 kD fusion protein was achieved by
the inducing of 1 mmol/L IPTG. At present, we are
carrying out to preparation antiserum using prokaryotic
expression vector of ASPV CP gene from Ya pear.
3 Materials and Methods
3.1 Virus source
Ya pear samples were collected from Korla pear in
Shayidong commercial orchard of Korla, Xinjiang,
China. This isolate was designed as Y. All these
symptomatic leaves and phloem were stored at 4
℃
.
3.2 Chemicals and reagents
Taq
DNA Polymerase was purchased from Gongdong
Dongsheng Biotech (China); dNTPs, Ribonuclease
inhibitor, IPTG were purchased from Shanghai
Sangon (China); M-MLV Reverse Transcriptase, T4
DNA ligase are from Fermentas (USA);
Nco
I,
Sal
I,
PMD19-T, DNA Marker D514A and Protein
Molecular Weight Marker D523A were all purchased
from TakaRa (China); TIANprep Mini Plasmid Kit
and TIANgel Midi purification Kit from TIANGEN
(China); others were all analysis purity made in China.
Expression vector PET-28a,
E. coli
DH5α and BL21
(DE3) as preserved strains were stored at
Biotechnology Laboratory of Horticultural Department,
Agriculture College, Shihezi University, China.
3.3 Primer design
The sequences were amplified by PCR reaction with
specific primers, which were designed according to
the cDNA sequence of ASPV from GenBank
NC_003462. Primer sequences are as follows: forward
primer (PS) 5’-CCCATTAGGTTAGGGTGTAGTTG
CT-3’ (nt 7829-7853) and the reverse primer (PA)
5’-ATGAAAGAAACACACACAT AGCCGC-3’ (nt
9249-9273). Forward primer (PS-E): 5’-CCATGG
ATGGCTTCCGATGGTTCG-3’ and the reverse
primer (PA-E) 5’-GTCGACC TTCCT GATTGATAA
AAC-3’ containing the
Nco
I and
Sal
I restriction sites
(underlined) containing the complete ORF of ASPV
CP gene according to the amplified sequence by the
primers PS and PA. All the primers were synthesized
by Shanghai Sangon Biotech and dissolved in
ultrapure water to 20 pmol/µL concentration for use.
3.4 Total RNA extraction and RT-PCR
Total RNAs were extracted from phloem infected by
Y using the approach described by He et al (2004).
The reverse transcription mixture contained 1.0 μL
specific reverse primer and 5.0 μL of total RNA and
9.5 μL of ddH
2
O. The mixture was kept at 70
℃
for 5
min, and then immediately transferred to ice for 5 min.
And then 2.5 μL of dNTPs (10 mM each), 5.0 μL of
5×M-MLV buffer, 1.0 μL of ribonuclease inhibitor (40
U·µL
-1
; Sangon, china), 1.0 μl of M-MLV reverse
transcriptase (200 U/µL; Fermentas, USA) and make
the total volume of 25.0 μL. The mixture was
incubated at 42
℃
for 1 h.
PCR reaction volumes were 20 μL, and contained 2.0 μL
of 10×PCR buffer, 0.5 μL of dNTPs (each 10 mM),
2.0 μL of forward and reverse primers, 2.0 μL of
cDNA, 0.2 μL (5U/µL)
Tap
DNA polymerase and
13.3 μL of ddH
2
O. PCR was carried out with an initial
denaturation of 4 min at 94
℃
, followed by 32 cycles
of 45 s, 94
℃
; 1min, 72
℃
; and then by a final
elongation step of 7 min at 72
℃
.
3.5 Cloning and sequencing
The amplified PCR products were gel purified and
extracted using TIANgel Midi Purification Kit