Using multiple-fragment amplification combined with Gibson assembly to clone genes with site-directed mutations / 生物工程学报

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.

Site-directed mutagenesis of long gene by partial amplification combining with double fragments ligation / 生物工程学报

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.

Study on immune effect of DNA vaccine pIRES2-MLAA34-HSP70 constructed by SOE-PCR / 重庆医学

Objective To construct the DNA vaccine pIRES2-MLAA34-HSP70,and to detect its immune effect.Methods The acute monocytic leukemia associated antigen gene MLAA-34 and heat-shock protein (HSP)70 gene were extracted by using RT-PCR.The specific overlapping primer was designed,and the fusion gene MLAA34-HSP70 was amplified by using SOE-PCR technique.Then the DNA vaccine pIRES2-MLAA34-HSP70 was constructed,and BALB/c mice were immunized with this DNA vaccine.The splenic lymphocyte killing activity was detected by using MTT,levels of IL-2,IL-4 and IFN-γ were also detected by using ELISA.Results The MLAA34-HSP70 gene (2 956 bp) and the DNA vaccine pIRES2-MLAA34-HSP70 was amplified and constructed successfully.The killing efficiency of DNA vaccine pIRES2-MLAA34-HSP70 in U937 cells was significantly higher than that in other experimental groups and control group (P＜0.01),and levels of IL-4,IL-2 and IFN-γin DNA vaccine pIRES2-MLAA34-HSP70 group were significantly higher than those in the other experimental groups and control group (P＜0.01).Conclnsion The DNA vaccine pIRES2-MLAA34-HSP70 is constructed successfully.It is shown that the DNA vaccine induces strong humoral immunity,which could enhance immune responses to tumor cells and specificlly kill MLAA34 positive cells.

An A-B Subunit Chimera of the E.Coli Verotoxin 1 Provides A Receptor Probe and Potential Vaccine Approach.

We have generated a chimera between the enzymatically active A subunit of the E coli derived AB5 verotoxin and a single receptor-binding B subunit. The construct was made by in frame fusion of the 3’ terminus of the A subunit gene with the 5’ end of the B subunit gene via the deletion of the intervening bases of the verotoxin operon such that the C terminus of the A subunit is continuous with the N terminus of a single B subunit. The gene product is a single fusion protein of 38kDa molecular weight, reactive with polyclonal and monoclonal antibodies against either the A or B subunits of the wild type toxin. The chimera showed a 104-105 fold reduction in cell vero cell cytotoxicity but no toxicity for the globotriaosyl ceramide (Gb3) deficient VRP subclone. No Gb3 binding by TLC overlay was detected. Polyclonal rabbit anti-VT1A-B chimera serum neutralizes VT1 cytotoxicity in vitro but reacts only with the A subunit of the wildtype holotoxin by western blot. This A-B chimera illustrates the importance of the pentameric B subunit in receptor binding and potentially identifies a novel attenuation vaccination strategy.

Codon optimization of 1, 3-propanediol oxidoreductase expression in escherichia coli and enzymatic properties

The gene dhaT from Klebsiella pneumoniae encoding 1,3-propanediol oxidoreductase (PDOR) was de novo synthesized by splicing overlap extension polymerase chain reaction (SOE-PCR) primarily according to Escherichia coli’s codon usage, as well as mRNA secondary structure. After optimization, Codon Adaptation Index (CAI) value was improved from 0.75 to 0.83, meanwhile energy of mRNA secondary structure was increased from -400.1 to -86.8 kcal/mol. This synthetic DNA was under control by phage T7 promoter in the expression vector pET-15b and transformed into the E. coli BL21 (DE3) strain. Inducers such as isopropyl beta-D-thiogalactoside (IPTG) and lactose were compared by activity at different inducing time. The activity of PDOR after codon optimized was 385.4 +/- 3.6 U/mL, which was almost 5-fold higher than wild type (82.3 +/- 1.5 U/ml) under the flask culture at 25ºC for 10 hrs. Then his-tagged enzyme was separated by using Ni-IDA column. The favorite environment for enzyme activity was at 5°C and pH 10.0, PDOR showed a certainly stability in potassium carbonate buffer for 2 hrs at diverse temperatures, enzyme activity was significantly improved by Mn2+.

Single-stranded megaprimer splicing through OE-PCR: Construction of full-length Aspergillus niger arginase cDNA.

A useful variant of PCR technique was devised to generate full-length Aspergillus niger arginase cDNA for expression. Briefly, a 450 bp amplicon was first constructed through overlap extension PCR (OE-PCR) by splicing in a 101 nucleotide long single-stranded megaprimer, facilitated by inclusion of an additional, shorter forward primer in the reaction. The amplicon was suitably cloned into pBlueScript to obtain pArg440 and the insert sequenced. The full-length arginase cDNA was subsequently assembled in pArg440 and moved into pET23a for heterologous expression. An interesting feature of this strategy was not to stoichiometrically incorporate the oligonucleotide megaprimer, but use it only as an early template. This OE-PCR strategy to utilize long single-stranded megaprimer may prove handy in terms of efficiency, yield and sequence choice.

Secretory-expression of Antimicrobial Peptide Bactenecin7 Gene in Lactococcus lactis and Analysis the Bioactivity of Its Expression Products / 中国生物工程杂志

To construct a secretory-expression vector of antimicrobial peptide Bactenecin 7(Bac7),and identify the secretory-expression product in L.lactis MG1363 and its bioactivity.The splicing primers of regulation elements and Bac7 gene,which designed according to codon usage preferences of L.lactis MG1363,were chemically synthesized,and the overlap-extension PCR method was used to splice the full length of Bac7 gene.Then the Bac7 gene was linked to expression vector pMG36e to construct pMG36e/Bac7 vector,and pMG36e/Bac7 was transformed into L.lactis MG1363 by electrophoration.RT-PCR and Western blot assays were applied to investigate the expression of the Bac7 gene in L.lactis,and bioactivity of Bac7 in culture supernatant of L.lactis was tested with plate-diffusion method.The results showed that the Bac7 gene and its regulation elements was amplified and cloned in the vector pMG36e successfully,The secretory-expressed Bac7 in L.lactis MG1363 harboring pMG36e/Bac7 was identified by Western blot,and it had high bacteriostatic activity against E.coli.These results indicate that the recombinant L.lactis MG1363 could express bioactive Bac7,which lays a foundation for further study of oral administration of a Bac7-secreting L.lactis to treat intestinal bacteria infection.

Construction and Enzyme-activity Assessment of L-Asparaginase Mutants / 中国药科大学学报

AIM:To construct nine novel L-asparaginase mutants and study their enzyme-activity.METHODS:The mutants were constructed using overlap extension PCR according to the principle of alanine-scanning mutagenesis. The enzyme-activity was detected by Nessler's method. RESULTS:The DNA sequencing showed that the mutagenesis was consistent with the theoretical prediction. The enzyme-activity assay demonstrated that each mutant possessed enzyme activity equal to the original enzyme. CONCLUSION:Through gene modification,epitop of L-asparaginase was changed without activity loss.These results provide foundation for further study of the structure-function relationship of L-asparaginase.