Construction of recombinant PRRSV ORF5 gene by DNA shuffling technique / 生物工程学报

Recombinant PRRSV △2ORF5 gene was constructed using DNA shuffling from four genetically different strains of PRRSV to study its heterologous cross-neutralizing ability. The △2ORF5 mutant gene was cloned into the vector pET-32a and transferred into E. coli BL21. SDS-PAGE confirmed that the molecular weight of the recombinant △2ORF5 was about 42 kDa, consistent with the predicted result. Then the purified recombinant protein was injected into BALB/c mouse to obtain polyclonal antibody. Western blotting analysis with mouse-anti-△2ORF5 polyclonal serum indicated that the parental virus recombinant GP5 protein reacted with the specific antibodies. Four parental viruses could be inhibited by the anti-△2ORF5 polyclonal antibody and the inhibition rates were higher than 53%. This work has laid a foundation for further development vaccine for PRRSV.

Protease gene shuffling and expression in Pichia pastoris

Four kinds of neutral and alkaline protease genes from Aspergillus oryzae and Bacillus subtilis were isolated and shuffled. The shuffled genes were selected, inserted into pGAPZαA plasmid and transformed into Escherichia coli. The gene which could express high-activity protease was selected by screening the sizes of transparent zones around the colonies on casein plates. After an ideal protease gene was selected, it was sequenced and then transformed into Pichia pastoris X33. The result showed that the base in 1022th position of shuffled protease gene was changed from thymine to cytosine, inferring that cysteine was changed to arginine in the mutant protease. After 48 h incubation for the transformed P. pastoris with the mutant or native protease genes, the mutant protease activity was 36.4% higher than the native protease (P<0.05). The optimal pH and temperature of the mutant protease were 6.5-8.0 and 30-70°C, respectively, which indicated better stability than the native protease (P<0.05).

The Progress and Application of Recombinant PCR / 中国生物工程杂志

Recombinant PCR applies to fulfill gene recombination by PCR thermal reaction.Over the twenty years,it has branched into three characteristic strategies:splicing by overlapping extension(SOE),jumping polymerase chain reaction(JPCR)and DNA shuffling.Recently,the technique aimed with exploiting natural source of different allele genes is developing up on simplification of experimental procedure,on trap for mutation and variation,and on highthroughput screening with technology of surface display and fluorescent probe.The recombinant PCR is increasesing value in broad range from biological basic research to bioengineering study.

DNA Shuffling of Vitreoscilla Hemoglobin / 中国生物工程杂志

To improve the growth enhancement activity of Vitreoscilla hemoglobin(VHb), Vitreoscilla hemoglobin gene(vgb) was mutated by error-prone PCR and then reconstituted by DNA shuffling. The shuffling library was constructed by inserting the shuffled genes into the downstream of vgb natural promoter and transforming them into E.coli DH5?. Mutated active VHb proteins were first screened in test tubes according to host cell pellets color and then in shake flasks according to host pellets wet weight .One active mutant protein, VHb′042506, was obtained after second screening. It could increased the host wet weight by 31.25% and 58.75% than that of the control which bearing natural VHb under microaerobic and extremely microaerobic conditions, respectively. Sequencing and alignment results showed that 11 nucleotides were mutated, thus resulted in 4 amino acids changes occurred in this mutant protein. CO difference spectrum test also indicated that it had higher specific absorption.

DNA Shuffling of Arabidopsis thalianna K+ Uptake Transporter Gene / 中国生物工程杂志

The DNA fragment sized 2 139bp, the same Sequence with AtKup1 gene from Arabidopsis thalianna was used as the templates for DNA family shuffling. The shuffeld AtKup1 gene library was expressed in the mutant of 5. cerevisae in which potassium transporter gene TRK1 and TRK2 were knocked out by homologous recombination. Then the screening was carried out in the low potassium media containing 5. 0 mmol/L KC1 and no histidine in it. it was found that both of diverse and wild AtKup1 gene can rescues the trk1△trk2△yeast mutant strain in low [ K + ] medium. The growth of 2 clones yeast containing diverse AtKup1 were beter than that of AtKup1 wild gene transformant. The sequencig results of the shuffeld AtKup1 showed that there were 2 nucleotide changed, which resulted in 2 amino acid variations in it compared with the original AtKup1. The potassium uptaking capacity of shuffled AtKup1 gene increased significantly when it was transformed into tobacco.

Affinity maturation of a single-chain antibody for hepatocellular carcinoma by error-prone PCR and DNA shuffling / 中国病理生理杂志

AIM: To obtain a single-chain antibody with high affinity for hepatocellular carcinoma (HCC). METHODS: A second single-chain antibody mutant library was established by using error-prone PCR and DNA shuffling. Single-chain antibodies with high affinity for hepatocellular carcinoma were selected from phage antibody library by using ELISA. RESULTS: The content of the second single-chain antibody mutant library was about 4.5?10~7. Two selected mutants M25 and M36 were obtained after 3 rounds of panning and ELISA. Immunoassay showed that M25 and M36 bound to human HCC cells specifically. The relative affinity of M25 was 2.0 folds higher than that of the original antibody, and M36 was 2.4 folds higher than the original antibody. CONCLUSION: Error-prone PCR combined with DNA shuffling is an effective method to improve affinity of antibodies isolated from phage antibody library.