3. Result
3.1 Expression of VP3
As shown in Fig. 2, four recombinant proteins expressed in E colisysterm including His-VP3, His-VP3-Tf16, His-VP3-NusA and TRX-His-VP3
were approximately 107 kDa, 107 kDa, 162 kDa, and 112 kDa, respectively.
Compared with His-VP3,after the addition of fusion protein and
molecular chaperone, the expression of VP3 in the supernatant increased.
Taking into account,the fusion protein needs to be purified again after
excised, resulting in loss of VP3. However, His-VP3-Tf would not have
such a concern, so pET-28a-VP3-pTf16-BL21 (DE3) was chosen to optimize
the expression conditions.
3.2 Optimization of expression
conditions
The pET-28a-VP3-pTf16-BL21 was selected for the VP3 expression and a
series of conditions optimization were carried out. There were 4 factors
in the optimization, including temperature, IPTG concentration,
L-arabinose concentration, and time. The results showed that VP3
expression was the highest in the supernatant when 0.3 mM of IPTG
(Figure 3b) and 3.5 mg/ml of L-arabinose (Figure 3c) were used to induce
expression at 20 ℃ (Figure 3a) for 12h (Figure 3d).
3.3 Purification and identification of
VP3
VP3 eluted at the 100mM of imidazole concentration, and all fractions
with high absorbance were collected and identified by SDS-PAGE and
Western-blot after concentrate. The result showed that the purified VP3
has a single band, with a purity estimated to be over 75% (Figure 4a).
Western blot analysis indicated that the VP3 reacted specifically with
Anti-6×His tag antibody (Solarbio, China) (Figure 4b). The concentration
of VP3 following purification from BL21 (DE3) cells is 0.32 mg/ml.
3.4 Characterization of the
VP3
The optimal coating concentration was 3.2 ng/μl determined by chessboard
method. The titer of VP3 antibodies can reach
1:1.28×105 measured by ELISA method (Figure 5). The
Dot-ELISA test showed that the purified VP3 could react with the serum
of immunized mice with inactivated BTV-1 virus (Figure 6). IFA tests
showed that the serum of immunized mice with VP3 expressed by E.
coli could react to that expressed by 293T cells produce fluorescence
(Figure. 7). It can be deduced that the VP3 expressed in this experiment
is not much different in structure from the eukaryotic VP3.