Difference between revisions of "Part:BBa K1228010"

Revision as of 03:39, 27 September 2013

The device secrete Rabies virus strain ERA glycoprotein in bacillus subtilis

This device can secrete the Rabies virus strain ERA glycoprotein.it contain the Promoter Pveg, RBS spoVG and SacB signal peptide for B.subtilis (BBa_K541501)and the coding sequence of Rabies virus strain ERA glycoprotein(BBa_K12280002 ,The RNA genome of the virus encodes five genes whose order is highly conserved. These genes code for nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and the viral RNA polymerase (L).The complete genome sequences range from 11,615 to 11,966 nt in length.”Because of containing the neutralizing epitopes which are the targets of vaccine-induced immunity, the glycoprotein can stimulate the organism to produce antibody against Rabies.)

Sequence and Features

RESULT

1. The recombinant strains which were trans formed with the plasmid pht-304 were inoculated into 5 ml LB broth containing 25μg erythromycin ml− 1, shaken at 37° C overnight, and then 100 ml LB medium supplemented with erythromycin was inoculated with 1 ml recombinant B.subtilis overnight culture. The cultures were grown at 37°C with shaking at 200 rpm for 10h,

2. The strains carrying only the pht-304 shuttle vector with no insert were used as controls.

3. The liquid culture was centrifuged at 10000× g for 10 min , and the cell-free supernatant was then concentrated with Millipore.

4. Add 20 ul 5XSDS sample buffer to 80 ul concentrated culture and heat 95°C for 10 minutes;

5. Load 20 μl onto 12% SDS-PAGE gel，80V for stacking gel , 120V for separation gel .

6. Make the gel for transfer in transfer buffer: 12V overnight, on ice.

7. Block the filter with blocking buffer for 1 hour at room temperature with gentle agitation on a platform shaker.

8. Discard blocking solution and immediately incubate filter with primary antibody.

9. Add 0.005 ml of primary antibody (1:5000) in to blocking solution. Incubate 2 hour at room temperature. with gentle agitation on a platform shaker.

10. Discard blocking solution and wash filter 3 times (5 minutes each time) with TBST.

11. Immediately incubate the filter with secondary antibody, add 0.003ml of secondary antibody solution (1:3000).

12. Incubate 1 – 2 hours at room temperature with gentle agitation.

13. Discard blocking solution and wash filter 3 times (5 minutes each time) with TBST.

14. Chemiluminescence for 1minute.

Rabies virus strain ERA glycoprotein successfully expressed in Bacillus subtilis,this is the picture of g-p western blot.

References

1. Morimoto K, Hooper DC, Spitsin S, et al. Pathogenicity of different rabies virus variants inversely correlates with apoptosis and rabies virus glycoprotein expression in infected primary neuron cultures. J Virol, 1999; 73, 510‐8. 2. http://en.wikipedia.org/wiki/Rabies_virus