Session 6: Functional Proteomics II

With the completion of sequencing many species’ entire genomes, we can easily clone genes, but the excellent vectors are requirement. The currently available vectors have some disadvantages: high-background, lowthroughput or non-directed cloning. High-background is generated by false-positive clones, which are almost caused by the recircularization of linearized vectors that have lost some bases at their ends due to digestion with contaminating exonuclease. To overcome these drawbacks, especially serve for high-throughput research, we have developed a zero background, high-throughput and directional or non-directed clone vector based on reporter gene reconstruction and regulatory element modulation. First, a mannase gene was amplified with a primer pair (F1:5 -AAA GTC GAC AAG GAG AGA TCT ATG GTC GTG ACT GGG AAA ACC CTG GCG ATG GGG GAG TTG CAT TTG TTT AAG A-3 ; R1:5 -AAA CAT ATG CTG GGT TAG GCG ATA TGA ATG CTT A-3 ) and cloned into pMD-18T directionally. E. coli X-gold was transformed and the recombinant plasmid was named pMD-18TM. Then, we synthesized another primer pair (F2:5 -AAA GAA TTC CCT TAG GCC AAA CCT CAC T TG GAT GGT GAG CAA GGG CGA GG-3 ; R2: 5 AAA GAT CT CCA AAC CTG AGG TGG ACG GGG GAG GGG CAA ACA A -3 ) to amplify a DNA fragment including egfp gene ORF flanking with two different Bsu36I and two XcmI digest sites. The PCR product was inserted into pMD-18TM directionally. The second recombinant plasmid was named pMD-18TMG. Because the two Bsu36I site have different recognized sequences, the plasmid can not be self-ligated after digested with Bsu36I and can be used for directional PCR cloning, alike XcmI for TA PCR cloning. In the Bsu36I cloning method, The extra sequences: 5 -TTACGAAGGAG-3 , and 5 TGAC-3 must be introduced into the 5 end of forward primer, the 5 end of reverse primer respectively in PCR, thus treated the PCR product with T4 DNA polymerase under adding dGTP, the PCR product formed stick ends can complement to the stick end of vector for directional cloning. In TA PCR cloning method, if an A adding at the 3 end of PCR product, it can complement to the both 3 end of the pMD-18TMG digested with XcmI, then TA cloning can be realized. When the foreign gene insert into the pMD-18TMG, the RBS sequence recovery the expression of mannase, which can hydrolyze polymannan. Due to mannase expressed, many hydrolysis halos can be detected and dedicate the probably positive clones at about 99% rate in the polymannan screening plate. The light green clones dedicate cells transformed plasmid no digested that cause gfp expression. The remainder white clones without hydrolysis halos are almost all negative clones generated by the recircularization of linearized vectors. We have used the vector to clone about one hundred cDNAs, and statistics dedicates that efficiency of clone is very high. 6.2 Threading Peptides, Proteins into Disease Surveillance Strategies