Constructing a phage-displayed random mutation library of HIV-1 Tat38-61 at the sites of 51 and 55 amino acids in basic region

Abstract:

We constructed a phage-displayed random mutation library of Tat38-61(51N/55N), for studying the molecular evolution screening of HIV-1 Tat38-61 epitope. We used primers containing the random nucleotide sequences, and introduced the random mutations at the sites of 51 and 55 amino acids coding sequences into full-length Tat sequences by overlapping PCR. With the randomly mutated full-length Tat as template, the Tat38-61(51N/55N) mutants which contained recognition sequences for the Xba I in both ends were amplified by PCR using the designed primers. The mutants were cloned into Xba I site in the phagemid vector pCANTAB5S, then the recombinants were transformed into E. coli TG1, a phage-displayed the random mutation library of Tat38-61(51N/55N) was constructed by the rescue of help virus M13KO7. The results showed that the library consisted of about 5.0×106 colonies and the phage library titer was 2.65×1012 TU/mL. More than 56.50% colonies in the library were positive for insertion. Sequence analysis showed that the nucleotides encoding amino acids at the sites of 51 and 55 distributed randomly. The constructed mutation library could meet the requirements for the following molecular evolution screening, and might prepare the Tat mutants for the further study of new Tat vaccine candidates.

Key words:HIV-1 Tat, basic region, random mutation, phage display