Preparation And Identification Of Liposome With H-2K~d/peptide Complex On The Surface

In this study, we attempt to cloning, expression and purification of mice H-2K~d,β2m elements. Using RT-PCR technology the full-length cDNA of H-2K~d extracellular region from SP2/0 cells was amplified. The fragment of H-2K~d was amplified by PCR and added 6-his-tag in the end of the carboxyl of H-2K~d. recombinant plasmid pET22b-H-2K~d was Successful constructed. Expression of H-2K~d andβ2m mainly exists in the form of inclusion bodies. H-2K~d andβ2m protein were purified by washing inclusion bodies respectively. H-2K~d was folded in the presence ofβ2m and a specific H-2K~d restrictive peptide of influenza virus to form H-2K~d -peptide complex, it was purified passing through a Sephacryl-300 column.Taking the dioleoylphatidylcholine (DOPC), cholesterol (CHOL) and nickel metal chelating phospholipid (1,2-dioleoyl-sn-glycero-3- [N-(5-amino-1- carboxypentyl) iminodiacetic acid] succinyl , DOGS-NTA-Ni) for lipid material of liposome, the film dispersion method was adopted to prepare the DOPC/DOGS-NTA-Ni liposome. And analysis characteristics of the liposomes shape distribution and particle size by dynamic laser light scattering instrument and transmit electron microscope. It was shown that its surface configuration was round and smooth, Average size of DOPC/DOGS-NTA-Ni liposome was 75nm.On the basis of above mentioned works, the H-2K~d-peptide complex and DOPC/DOGS-NTA-Ni liposome were mixed at a ratio of 2:1 and were incubated at 4℃overnight. We desired the H-2K~d-peptide complext containing 6-his-tag at the end of the carboxyl could display on the liposome surface. Particle size was observed when H-2K~d-peptide complex display on the liposome surface than pro-liposome by dynamic laser light scattering instrument, the average size was came to 94 nm from 75nm. And TEM observations found that H-2K~d-peptide complex was display on the surface of DOPC/DOGS-NTA-Ni liposome.Our date demonstrate that we had successfully display H-2K~d-peptide complex on the surface of liposome, which is the foundation for construction of artifical APC based on liposome.