Difference between revisions of "Part:BBa K2748000"

Revision as of 17:44, 17 October 2018

U24 protein from human herpesvirus 6A

U24 protein from human herpesvirus 6A is a C-terminal membrane anchored protein that can downregulate T cell receptors via endosomal recycling inhibition.

Usage and Biology

U24 is a small (87aa) tail-anchored protein(Sullivan and Coscoy, 2010) that can downregulate TCR/CD3 complex from the cell surface by exclusion of CD3 from Rab11-containing recycling endosomes and thus inhibiting TCR complex recycling back to the surface(Sullivan and Coscoy, 2008). While it was demonstrated later that U24 also downregulate transferrin(Sullivan and Coscoy, 2010), its action is relatively specific, without affecting the surface level of ICAM-1, MHC class I, ULBP1, ULBP2, CD4 and CD8(Sullivan and Coscoy, 2008). Since U24 does not colocalized with CD3, it is believed that the downregulation does not rely on the interaction of U24 with CD3 but instead results from interaction of U24 and the endosomal recycling machinery(Sullivan and Coscoy, 2010). In addition, unlike proteins from other herpesviruses that downregulate TCRs or B cell receptors (BCRs), U24 does not activate lymphocyte signaling pathways(Sullivan and Coscoy, 2008). Furthermore, it was demonstrated that U24 can impair T cell activation by antigen presenting cells(Sullivan and Coscoy, 2008).

Design Considerations

The nucleotide sequence of U24 coding sequence was retrieved from NCBI nucleotide database(NCBI Reference Sequence: NM_002006.4:465-932), and synthesized by IGE Biotechnology LTD. Biobrick prefix and suffix was added by PCR using the following primers,

U24-prefix: 5’ cggaattcgcggccgcttctagATGGATCCCCCTCGGACGC 3’

U24-suffix: 5’ AActgcagcggccgctactagtaTCATCGCCTTTGACGATTCACAT 3’

and ligated onto the pSB1C3 plasmid backbone obtained from digestion of pSB1C3-lacI-RFP(Part:BBa_J04450). The sequence was comfirmed by Sanger sequencing by IGE Biotechnology LTD using VF2 as the forward primer.

Note that this part comes in the absence of the stop codon at the end of the sequence, hence this part should be cloned into vectors with pre-existing stop codon, fused with other protein with stop codon, or added a stop codon using PCR prior to use.

Results

For more details, please check out our [http://2018.igem.org/Team:SYSU-CHINA#/Demonstrate result page]!

In iGEM 2018, SYSU-CHINA attempted to develop a reversible safe switch for CAR T therapy based on the tet-inducible CMV promoter and U24 protein of Human Herpesvirus 6. Since it is the major part of our project, we conducted extensive research on U24 and obtained valuable results..

Figure 2: Fluorescence and western blot analysis of U24 expression under different concentration of doxycycline

The transgenic BMSCs was cultured for 48 hours and the supernatant, theoratically containing certain growth factors, was collected. Scratch wound healing assays were performed using HEK293T cells with the collected supernatant. The data below suggested that bFGF produced by transgenic BMSCs is able to accelerate wound healing.

Figure 7: Results of scratch wound healing assays

To further demonstrate the potentials of the bFGF-transgenic BMSCs, Collected supernatant mentioned above was used to culture uterine endometrium cells, and MTT assay were performed after 48 hours. The result indicated that bFGF secreted by engineered BMSCs is able to promote endometrium cell growth.

Figure 8: Results of MTT assays

References

Sullivan, B.M., and Coscoy, L. (2008). Downregulation of the T-cell receptor complex and impairment of T-cell activation by human herpesvirus 6 u24 protein. Journal of virology 82, 602-608.

Sullivan, B.M., and Coscoy, L. (2010). The U24 protein from human herpesvirus 6 and 7 affects endocytic recycling. Journal of virology 84, 1265-1275.

Sequence and Features