Site-directed Mutagenesis Based on Overlap Extension PCR

[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension PCR for twice obtained the mutation gene which of the full length of the recombinant Human Tissue type plasminogen activator （Reteplase）. The mutation gene cloned it into pEASY- blunt simple cloning vector for sequencing. [Result] The sequencing results showed that three site mutations were fully consistent with the expected results （10~ site had been added a base-pair of A, C had been changed into G at 137~ site, G had been changed into A at 686~ site）.Three site mutations were introduced by using overlap extension PCR on one-step. The overall rate of obtaining the mutant sites was 100%. Site-directed mutagenesis will clone the recombinant Human Tissue type plas- minogen activator and laid the basis for the functional study. [Conclusion] Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is an efficient, convenient and economical DNA-directed mutagenesis method.

Artificial Synthesis of TAT PTD-Tachyplesin Fusion Gene by Overlap Extension PCR

This study aimed to investigate the synergism of the TAT PTD （ protein transduction domain in HIV-1 transactivator of transcription protein） to antibacte- rial peptide tachyplesin from Tachp/eus tr/dentatus. Treated with Pichia pastoris preferred codon optimization, using the eDNA sequence of tuchyplesin mature pep- tide （54 aa） harboring TAT FFD sequence （11 aa） as reference template, six single-stranded oligonueleotides were designed, the sequences of restriction sites EcoR I and Xba I were introduced to the 5＇ end of primers P1 and P6, respectively. TAT PTD ＋ Tachyplesin fusion gene with a full length of 219 bp was artificially synthesized by overlap extension PCR, which laid the preliminary foundation for subsequent functional and synergism studies.

Site-directed Mutagenesis of Streptomyces avermitilis aveD Gene

[Objective] The aim of this study was to produce Streptomyces avermitilis strain with site-directed mutagenesis in aveD gene,and so as to provide theoretical basis for genetic breeding of S.avermitilis.[Method] PCR-driven overlap extension was conducted for the site-directed mutagenesis in aveD gene;the mutated aveD gene then was used to construct vector pDC3（pKC1139∷aveD） via molecular manipulations like in vitro enzyme digestion and ligation;the vector pDC3（pKC1139∷aveD） was then introduced to aveD deletion mutant 489 of avermectin-producing strain S.avermitilis 76-9.[Result] Mutant strain 536 of site-directed mutagenesis of S.avermitilis 76-9 was obtained by homologous recombination.The sequencing results show that the sixty-ninth base C in aveD-coding region of mutant 536 was substituted by T,and the corresponding amino acid Thr was mutated to be Ile.[Conclusion] This study laid basis for the development of strains specifically producing avermectin B.

Total Chemical Synthesis, Assembly of Human Torque Teno Virus Genome

Torque teno virus(TTV) is a nonenveloped virus containing a single-stranded,circular DNA genome of approximately 3.8kb.We completely synthesized the 3 808 nucleotides of the TTV(SANBAN isolate) genome,which contains a hairpin structure and a GC-rich region.More than 100 overlapping oligonucleotides were chemically synthesized and assembled by polymerase chain assembly reaction(PCA),and the synthesis was completed with splicing by overlap extension(SOEing).This study establishes the methodological basis of the chemical synthesis of a viral genome for use as a live attenuated vaccine or gene therapy vector.

Activity after Site-Directed Mutagenesis of CD59 on Complement-Mediated Cytolysis

CD59 may inhibit the cytolytic activity of complement by binding to C8/C9 and protect host cell membranes against homologous membrane attack complex （MAC）. However, CD59 is widely overexpressed on tumor cells, which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is still confused. As reported the MAC binding site is located in the vicinity of a hydrophobic groove on the membrane distal face of the protein centered around residue W40. Here two site-directed mutagenesis were performed by overlapping extension PCR to delete residue W40 site （Mutant 1, M1） or to change C39W40K41 to W39W40W41 （Mutant 2, M2）. Then we constructed mutant CD59 eukaryotic expression system and investigated their biological function on CI-IO cells compared with wild-type CD59. Stable populations of CHO cells expressing recombinant proteins were screened by immunotechnique. After 30 passages culturing, proteins could be tested. Dye release assays suggest that M1CD59 loses the activity against complement, while M2CD59 increases the anti-complement activity slightly. Results indicate that W40 of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and to treat tumors. Cellular ＆ Molecular Immunology.