| HIV-1 vaccine is one of the most difficult human vaccine to overcome. The existing vaccine antigens can not induce broadly and high effective neutralizing antibody, which is a major obstacle to the effective vaccine design. HIV-1 whole membrane protein, recombinant protein and virus-like particles, there are some disadvantages such as low exposure level, unspecific epitope competition, which can not be an ideal vaccine candidate antigen. HIV-1 envelope protein gp41 MPER is capable of causing a wide variety of important targets for neutralizing antibodies(2F5 and 4E10 and 10E8), MPER region plays an important role in the fusion process. Gp120 bingding with CD4, then MPER region occurred a series of conformational changes. Neutralizing Antigen epitope also had a transient exposure, by the body’s immune system recognition, resulting in a broadly neutralizing antibody response. But the complex and variable conformation of MPER region prevent us to know the detailed information of conformation, so we did the following design in order to obtain a broadly neutralizing antibody response to the MPER region:First, we used the random mutation PCR technology to construct a gp41-MPER mutant library, and then the mutant library were constructed to the expression vector p CTCON2, ultimately form a p CTCON2-MPER mutation library. In order to make the library to produce a sufficient degree of mutation, not only to make a certain change of protein structure, but also not too much change. Error-Prone PCR conditions 0.2% to 5% rate error will be used.Second, using the yeast surface display system, the mutant library will be transferred into the yeast cells, glucose switch to galactose to induced expression, which will display the mutant protein library to the yeast surface.Third, using flow cytometry screening technology was used to screen mutant strains which have high affinity with neutralizing antibody 2F5 and 4E10, the selected cells back into the culture medium overnight, repeat this procedure again, secondary screening, after screening for four times,we can obtained that the obtained the strongest binding capacity of antigen displaying yeast strains with the neutralizing antibody 2F5 and 4E10.Fourth, sequence analysis of selected antigens was performed to determine the target antigen.To extract the genome and determine the location of the mutation.Analysis of the ability to induce neutralizing antibodies against antigens, we immunized guinea pigs with the yeast, three immune, immune serum taken by indirect ELISA detection method, detection the immunogenicity of mutant protein antigen, the antibody titer of 4E10 and 2F5 like antibody in the serum of guinea pig. Detection of neutralizing antibody titers in guinea pig serum by using TZM-b1 cells as a pseudotype virus neutralization test.The significance of this study:(1)Using the yeast display technology to display the complex protein antigens which we design, high-throμghput screening the sequence and conformation mutant proteins.It is possible to obtain which site affect the degree of exposure of the known epitopes in space, and their whether influence the ability of antigen epitope binding neutralizing antibodies and the ability to induce neutralizing antibodies.(2)We selected mutated protein using yeast surface display system to obtain the correct folding and keep the natural conformation of antigen, screening the high affinity antigen can successfully induced broadly and effective neutralizing antibodies.(3)The ideal antigen structure analysis, analysis the similarities and differences of the mutation antigen on the structure and sequence with the original antigen, and wants to get large amounts of data information about the vaccine antigens design, to guide it as a candidate vaccine antigen. |
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