Study On Preparation And Determination Of Triple Conjugates: Cpps-Antibody-Albuminnanospheres

Objective: To explore the feasibility of applying N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) conjugation technology to the preparation of TAT-EGFP -Antibody-Albumin Nanospheres (TAT-EGFP-IgG-BSA-NS) with penetrating peptide (TAT-EGFP), antibody (HBSAg-IgG) and albumin nanospheres (BSA-Ns). And to identify the chemical composition, the coupling mode and the penetrating activity of the triple conjugates.To provide a basis for further studies on construction of a new drug delivery system: CCPs-Specific Antibody-Nanospheres conjugates(drug coated) to achieve the effects of security, targeting, specificity and efficiency to tumor cells.Methods: 1. Expression and determination of fusion protein TAT-EGFP: Recombinant strain BL21 was revived in order to extract recombinant plasmid of Pet30a-TAT-EGFP to sequence reference to the early experiments by our group; fusion protein TAT-EGFP was purified by affinity chromatography method of Ni-NTA Super flow Cartvidge. SDS- polyacrylamide gel electrophoresis (SDS-PAGE) assay was used to identify its purity and molecular weight; TAT-EGFP was incubated with EJ cells (bladder cancer cells) to detect the penetrating activity of TAT-EGFP with fluorescence microscope. 2. Preparation and identification of CCPs-Nanospheres conjugates: CCPs-Nanospheres conjugates (TAT-EGFP-BSA-NS) was prepared applying cross-linking agent of SPDP. SDS-PAGE assay was used to identify the chemical composition and the coupling mode of CCPs-Nanospheres conjugates. CCPs-Nanospheres conjugates was incubated with EJ cells to detect its penetrating activity with fluorescence microscope. 3. Preparation of the triple conjugates of CPPs- Antibody- Albumin Nanospheres: The method was the same as 2. 4. Identification of the triple conjugates: SDS-PAGE assay, Native-Page assay, indirect immunefluorescent assays and laser tweezers Raman spectroscopy (LTRS) were used to identify the chemical composition of the triple conjugates. The triple conjugates was incubated with EJ cells to detect its penetrating activity with fluorescence microscope.Results: 1. Expression and identification of TAT-EGFP: the sequencing results of the amplified TAT-EGFP gene was consistent with the expected targer sequence; the molecular weight of TAT-EGFP was 31Kd which SDS-PAGE showed; the concentration of TAT-EGFP was 14.2mg/ml in BCA assay; green fluorescent was seen in EJ cells which incubated with TAT-EGFP for 1 hour at excitation wavelength of 450nm-490nm under the fluorescence microscope. 2. Preparation and identification of CCPs-Nanospheres conjugates: one visible protein bands were showed at low molecular weight end under reducing SDS-PAGE, while no band was showed under non-reducing SDS-PAGE; green fluorescent was seen in EJ cells which incubated with TAT-EGFP-BSA-NS for 1 hour at excitation wavelength of 450nm-490nm under fluorescence microscope. 3. Preparation and characterization of CPPs-Antibody-Albumin Nanospheres : three visible protein bands were showed at low molecular weight end under reducing SDS-PAGE , while no band was showed under non-reducing SDS-PAGE; the sample bottom was white with no band seen ran into gel under Native-PAGEgel in the UV gel imager Text; a number of peak changes could be found in triple conjugates by LTRS which corresponding to the peaks of TAT-EGFP and IgG compared with nanospheres; the surface of the triple conjugates could be found visible green and red fluorescence under corresponding excitation wavelengths of fluorescence microscope; green fluorescent was seen in EJ cells which incubated with TAT-EGFP-IgG-BSA-NS for 1 hour at excitation wavelength of 450nm-490nm under the fluorescence microscope.Conclusion:The conjunction of cell-penetrating peptides, antibodies (IgG) and albumin nanospheres by the method of SPDP conjugation technology is successful, and it is confirmed that the triple conjugates of CPPs-Antibody- Albumin Nanospheres has the activity of penetrating EJ cells. Our research provide a basis for further studies on construction of a new drug delivery system: CCPs-Specific Antibody-Nanospheres conjugates (drug coated) to achieve the effects of security, targeting, specificity and efficiency to tumor cells.It is the ideal and feasible conjugation scheme that TAT-EGFP and albumin nanospheres are connected to IgG (as a connecting carrier) applying SPDP conjugation technology.The application of LTRS is firstly used d to identify the composition of protein conjugation, and the results of LTRS is the same as the results of other classical methods, such as SDS-PAGE, Native-PAGE and indirect immune- fluorescencet. It is confirmed that the identification of protein conjugates by LTRS is feasible.