Difference between revisions of "Part:BBa K1228010"

 
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<partinfo>BBa_K1228010 short</partinfo>
 
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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).
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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.)
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<partinfo>BBa_K1228010 parameters</partinfo>
 
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== RESULT ==
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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,
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2. The strains carrying only the pht-304 shuttle vector with no insert were used as controls.
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3. The liquid culture was centrifuged at 10000× g for 10 min , and the cell-free supernatant was then concentrated with Millipore.
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4. Add 20 ul 5XSDS sample buffer to 80 ul concentrated culture and heat 95°C for 10 minutes;
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5. Load 20 μl onto 12% SDS-PAGE gel，80V for stacking gel , 120V for separation gel .
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6. Make the gel for transfer in transfer buffer:  12V  overnight, on ice.
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7. Block the filter with blocking buffer for 1 hour at room temperature with gentle agitation on a platform shaker.
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8. Discard blocking solution and immediately incubate filter with primary antibody. 
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9. Add 0.005 ml of primary antibody (1:5000)  in to blocking solution. Incubate 2 hour at room temperature. with gentle
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agitation on a platform shaker.
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10. Discard blocking solution and wash filter 3 times (5 minutes each time) with TBST.
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11. Immediately incubate the filter with secondary antibody, add 0.003ml of secondary antibody solution (1:3000).
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12. Incubate 1 – 2 hours at room temperature with gentle agitation.
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13. Discard blocking solution and wash filter 3 times (5 minutes each time) with TBST.
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14. Chemiluminescence for 1minute.
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https://static.igem.org/mediawiki/igem.org/9/96/G-P--Western_Blot.png
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Rabies virus strain ERA glycoprotein successfully expressed in Bacillus subtilis,this is the picture of g-p western blot.
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== Discussion and future work ==
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In our project, glycoprotein has been successfully secreted by ''Bacillus subtilis''. Our final goal is to active immunity system of dogs through this vaccine. We will inoculate mice the vaccine in future work.
 +
Two kinds of express systems can be used for ''B.subtilis'' as antigen vector. We will try to display glycoprotein in the surface of Spore of ''B.subtilis'' through Chromosomal-encoded way in the future.
 +
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== Acknowledge ==
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Associate professor MinCui provided the glycoprotein coding gene for us. We are grateful to her. The following is her homepage. http://my.hzau.edu.cn/Article/ShowArticle.asp?ArticleID=1831
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== References ==
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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.
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2. http://en.wikipedia.org/wiki/Rabies_virus

Latest revision as of 10:56, 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 737
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1565
    Illegal BsaI site found at 1584
    Illegal BsaI.rc site found at 225
    Illegal BsaI.rc site found at 1791


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. G-P--Western_Blot.png

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


Discussion and future work

In our project, glycoprotein has been successfully secreted by Bacillus subtilis. Our final goal is to active immunity system of dogs through this vaccine. We will inoculate mice the vaccine in future work. Two kinds of express systems can be used for B.subtilis as antigen vector. We will try to display glycoprotein in the surface of Spore of B.subtilis through Chromosomal-encoded way in the future.

Acknowledge

Associate professor MinCui provided the glycoprotein coding gene for us. We are grateful to her. The following is her homepage. http://my.hzau.edu.cn/Article/ShowArticle.asp?ArticleID=1831


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