Difference between revisions of "Part:BBa K1071008"
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===Usage and Biology===
 
===Usage and Biology===
  
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To challenge PHAECTORY for the efficient antibody production and secretion we constructed an expression vector consisting of the following parts: <html><a href="http://2013.igem.org/Team:Marburg/Parts">BBa_K1071001-BBa_K1071008</a></html> and transfected them into the algae ''Phaeodactylum tricornutum''. A gene, which encoded for the Hepatitis B antibody, was placed under the control of a nitrate induciable promoter. Five different clones of PHAECTORY were grown to an optical density of 0.4 in a nitrate-containing medium (Figure). In a next step, we wanted to analyse how much Hepatitis B antibody was secreted from the algae into the surrounding medium. Therefore, intact cells were separated from the surrounding medium by centrifugation. Both, cell pellets and supernatant were analysed for the presence of Hepatitis B antibodies by Western blot analysis.
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To challenge PHAECTORY for the efficient antibody production and secretion we constructed an expression vector consisting of the following parts: <html><a href="http://2013.igem.org/Team:Marburg/Parts">BBa_K1071001-BBa_K1071008</a></html> and transfected them into the algae ''Phaeodactylum tricornutum''. Two genes, which encode for the Hepatitis B antibody, was placed under the control of a nitrate induciable promoter. Five different clones of PHAECTORY were grown to an optical density of 0.4 in a nitrate-containing medium (Figure). In a next step, we wanted to analyse how much Hepatitis B antibody was secreted from the algae into the surrounding medium. Therefore, intact cells were separated from the surrounding medium by centrifugation. Both, cell pellets and supernatant were analysed for the presence of Hepatitis B antibodies by Western blot analysis.
  
 
The Western blot analysis (Figure) shows that huge amounts of Hepatitis B antibodies were secreted into the medium. The positive signal in the cell pellet shows that antibody production by PHAECTORY was still in progress. Taken together, the amount of Hepatitis B antibodies in supernatant was significantly higher in the supernatant then in the cell pellet. This is the ‘proof of concept’ that PHAECTORY has not only the ability to produce high-value proteins (e.g. antibodies), but also secrets them in huge amounts into the surrounding medium. Therefore, PHAECTORY allows direct secretion of high-value proteins into the medium, and allows their easy purification with low effort and costs.
 
The Western blot analysis (Figure) shows that huge amounts of Hepatitis B antibodies were secreted into the medium. The positive signal in the cell pellet shows that antibody production by PHAECTORY was still in progress. Taken together, the amount of Hepatitis B antibodies in supernatant was significantly higher in the supernatant then in the cell pellet. This is the ‘proof of concept’ that PHAECTORY has not only the ability to produce high-value proteins (e.g. antibodies), but also secrets them in huge amounts into the surrounding medium. Therefore, PHAECTORY allows direct secretion of high-value proteins into the medium, and allows their easy purification with low effort and costs.
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==MIT_MAHE 2020==
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'''Summary'''
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The human monoclonal antibody IgG1 Cla4mAb recognises the Hepatitis B surface antigen (HBsAg) subtype adr, a protein present on the surface of the hepatitis B virus. Anti-HBs appears after convalescence from acute infection and lasts for many years. It can also be produced in response to hepatitis B vaccination.
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==References==
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<p>1. Maynard JE. et al. In Zuckermann AJ. (ed), Viral Hepatitis and Liver Disease. New York: Alan R. Liss Inc.; 1988; 967-969. Beasley RP, Hwang L. In Vyas GN. (ed), Viral Hepatitis and Liver Disease. New York: Grune & Stratton; 1984; 209-224. Specter S. Hepatitis B Vaccines. In Specter S. (ed), Viral Hepatitis, Diagnosis, Therapy and Prevention. Totowa, NJ: Humana Press; 1999; 377-391.</p>
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<p>2. Liang T. J. (2009). Hepatitis B: the virus and disease. Hepatology (Baltimore, Md.), 49(5 Suppl), S13–S21. https://doi.org/10.1002/hep.22881 </p>
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"
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1071008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1071008 SequenceAndFeatures</partinfo>

Latest revision as of 18:36, 23 October 2020

Hep B Antibody heavy chain

The human monoclonal antibody IgG1 Cla4mAb recognises the Hepatitis B surface antigen (HBsAg) subtype adr. This is the ORF for the heavy chain of the antibody (Origin: AF027159.2). The codon usage was adapted to Phaeodactylum tricornutum.

Usage and Biology

To challenge PHAECTORY for the efficient antibody production and secretion we constructed an expression vector consisting of the following parts: BBa_K1071001-BBa_K1071008 and transfected them into the algae Phaeodactylum tricornutum. Two genes, which encode for the Hepatitis B antibody, was placed under the control of a nitrate induciable promoter. Five different clones of PHAECTORY were grown to an optical density of 0.4 in a nitrate-containing medium (Figure). In a next step, we wanted to analyse how much Hepatitis B antibody was secreted from the algae into the surrounding medium. Therefore, intact cells were separated from the surrounding medium by centrifugation. Both, cell pellets and supernatant were analysed for the presence of Hepatitis B antibodies by Western blot analysis.

The Western blot analysis (Figure) shows that huge amounts of Hepatitis B antibodies were secreted into the medium. The positive signal in the cell pellet shows that antibody production by PHAECTORY was still in progress. Taken together, the amount of Hepatitis B antibodies in supernatant was significantly higher in the supernatant then in the cell pellet. This is the ‘proof of concept’ that PHAECTORY has not only the ability to produce high-value proteins (e.g. antibodies), but also secrets them in huge amounts into the surrounding medium. Therefore, PHAECTORY allows direct secretion of high-value proteins into the medium, and allows their easy purification with low effort and costs.

Secretion

MIT_MAHE 2020

Summary

The human monoclonal antibody IgG1 Cla4mAb recognises the Hepatitis B surface antigen (HBsAg) subtype adr, a protein present on the surface of the hepatitis B virus. Anti-HBs appears after convalescence from acute infection and lasts for many years. It can also be produced in response to hepatitis B vaccination.

References

1. Maynard JE. et al. In Zuckermann AJ. (ed), Viral Hepatitis and Liver Disease. New York: Alan R. Liss Inc.; 1988; 967-969. Beasley RP, Hwang L. In Vyas GN. (ed), Viral Hepatitis and Liver Disease. New York: Grune & Stratton; 1984; 209-224. Specter S. Hepatitis B Vaccines. In Specter S. (ed), Viral Hepatitis, Diagnosis, Therapy and Prevention. Totowa, NJ: Humana Press; 1999; 377-391.

2. Liang T. J. (2009). Hepatitis B: the virus and disease. Hepatology (Baltimore, Md.), 49(5 Suppl), S13–S21. https://doi.org/10.1002/hep.22881

" Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 516
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1026
    Illegal SapI.rc site found at 1374