Page 5 - BM 2010 Vol.1 No.2

Bioscience Methods
BM 2010, Vol.1, No.2 http://bm.sophiapublisher.com
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and its insecticidal activity was greater than that of
known HD-73 strains (Xie et al., 2010).
Bt W015-1 strains can synthesize crystalline
inclusions during sporulation with a molecular mass
of 133 kD. The crystal protein possesses different
restriction enzymatic digesting sites compared to that
of Bt strain HD-73.
In this research we constructed the prokaryotic
expression vector, pQE30-Cry1Ac22, to heterologously
express in Eescherichia coli M15 and purified the
inclusion protein His-tag-Cry1Ac22 in order to
understand the characteristics and functions of the
CryAc22 insecticidal crystal protein.
1 Results and analysis
1.1 The construction and identification of pQE30-
Cry1Ac22 prokaryotic expression vector
We amplified the Cry1Ac22 gene, in the length of
about 3 500 bp, from the plasmid DNA of Bt strain
W015-1 ligated to the sequencing vector named as
pMD18-T-Cry1Ac22 which was identified by the
restriction enzymes BamHⅠ and Sal Ⅰ (Figure 1A).
Sequencing analysis confirmed the amplified gene to
be nearly identical to the Cry1Ac22 deposited in the
GenBank. We ligated the gene from the pMD18-
T-Cry1Ac22 into the prokaryotic expression plasmid
pQE30 to construct E.coli expression vector by cutting
with the restriction enzyme BamHⅠ and SaIⅠ (Figure
1B). Further, the recombinant plasmid was validated
by the restriction enzyme BamHⅠ and named
pQE30-Cry1Ac22 (Figure 1B).
The targeted gene cry1Ac22 in the recombinant has a
length of about 3.5 kb, while its vector pQE30 has a
length of 3.4 kb. It is too similar in length to be
distinguished by digestion with BamHⅠ and SaIⅠ
followed by agarose electrophoresis. Instead, we used
a single restriction enzyme, BamHⅠ, to cut the
recombinant to obtain a single band about 6.9 kb in
length.
The pQE30-Cry1Ac22 plasmids were transformed
into competent cells of E.coli M15 and single positive
colonies were identified by PCR (Figure 2). Figure 2
shows that lane 1 to lane 5 were positive transfor-
mants which confirmed that the recombinant plasmid
had been transformed into host bacterial cells of E.coli.
The transformed Escherichia coli harboring plasmid
pQE30-Cry1Ac22 was induced with 1 mmol/L IPTG
to produce the recombinant proteins and 7.5℅
polyacrylamide gel electrophoresis was performed on
the induced lysate to identify the inclusion protein.
The results of SDS-PAGE showed that a distinct band
of about 133 kD in molecular weight existed in the
induced bacteria lygate transformed with pQE30-
Cry1Ac22, whereas the band was lacking in the
control without induction (Figure 3). The results
indicated that recombinant plasmid pQE30-Cry1Ac22
should produce fusion protein 6× His-Cry1Ac22 under
the induction of IPTG, and that the expressed
amount of the fusion protein was increased gradually
with the increase of inducing time (Figure 3).
In order to stabilize harvest of the expressed fusion
proteins, we optimized the conditions for expressing
the fusion proteins under temperature at 21 ℃ , 28℃
and 37 ℃ and concentrations of IPTG at 0.1 mmol/L,
0.5 mmol/L and 1.0 mmol/L. The optimum condition
was identified as 1.0 mmol/L IPTG concent ration at
Figure 1 Construction and identification of pQE30-Cry1Ac22
Note: M: λDNA/HindIII marker; A: pMD18-T-Cry1Ac22 digested with BamHⅠand SalⅠ; 1~2: pMD18-T-Cry1Ac22; B: pQE30
digested with Bam HⅠand SalⅠ; 1~2: pQE30; C: pQE30-Cry1Ac22 digested with BamHⅠ; 1~2: pQE30-Cry1Ac22