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Objective: various conserved motifs by site-directed mutagenesis techniques mutations in the body (p201 / a the p20s 1 / a p20k / a, p20i / a p20v 1 / a, p20v 2 / a), and by Western blotting explore conserved motifs in the amino acids of the rat mucin rMuc3 protease cleavage impact. Method: 1, the use of site-directed mutagenesis techniques, primer design corresponding mutation based on PCR amplification mutant p20 as a template, and verified by sequencing. 2, with the amount of plasmid extraction kit to obtain a sufficient amount of plasmid, and then using the cationic liposome LipofectAMINE mutant p20 was transfected into COS-7 cells. 3, through the expression of the Western blotting analysis of each mutant, using Quantity one analysis of various mutants not digested and the digestion portion of the expression intensity and analysis part of the proportion is not digested. Results: 1, using PCR-directed mutagenesis, mutant clustalx1.83 software template p20 than after sequencing confirmed the mutation completely successful. And by the amount of plasmid was extracted to obtain a sufficient amount of plasmid used for transfection. 2, after cell lysis, the cell lysate the Western blotting immunoblot experiments, confirmed to exist in the 55 kDa at the portion not digested, may be in the presence of 30kDa at the N-terminal portion of the anti-V5 antibody detected after found in Quantityone analysis, S < sub> 2 / A completely inhibited the occurrence of the digested, G / A inhibition of the vast majority of the DNA portion accounting for 79% (not digested), L / A, the I / A, V 1 / A, V 2 / A different degree of inhibition of digestion, not digested were 22%, 39%, 14% and 17%, respectively, while the K / A and S 1 / A of digested almost no effect, not digested part of 6% and 3%, respectively, the Cleavage Efficiency and p20 (not digested partially accounted for 4%) is almost the same as . Conclusion: p20 as a template for various mutants mutation successful liposome transfection method, all mutant into COS-7 cells and efficient expression of the corresponding mutant. By Western blotting immunoblotting experimental results reveal the the digestion conserved motifs LS 1 KGS 2 IV 1 V 2 in which each amino acid protease cleavage occurs is very important, and the mechanism may be because this conserved motif is located in the annular region between β2 and β3 slice, the conformation of the amino acid to maintain mucin is a must. S 1 O-sugar chain, and removal of O-sugar chain does not affect the digested conserved motifs LS 1 the KGS 2 < / sub> IV 1 V 2 digested.
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