| P-lactam antibiotics, can be used for disinfection by inhibiting the synthesis of bacterial cell wall and destroying the formed cell wall. Currently,β-lactam antibiotics'global sales have reached 15 billion U.S. dollars, which is 65% of the semisynthetic antibiotics. Among these antibiotics, Penicillins and Cephalosporins are the dominant products. With advantages such as low toxicity, acid resistance and multiple modification sites, cephalosporins are increasingly becoming the preferable antibiotics.Cephalosporin C (CPC) was used as raw material, and it was first transferred to 7-Amino Cephalosporanic Acid (7-ACA), then chemically modified to semi-synthetic, Cephalosporins was synthesized in following this process. As a metabolite of Acremonium chrysogenum, CPC was produced by deacetylation of cephalosporin C (DAC) with acetyltransferase which was the last reaction of its biosynthesis and was also the rate-limiting step for bio-CPC synthesis. It's proved that the low reaction rate was due to the deficiency of acetyltransferase. Currently, the research about acetyltransferase and its encoding gene- cefG was rarely reported.According to NCBI, the cefG gene of Acremonium chrysogenum can not be replicated from genome directly because there are two introns in its DNA sequence. In this paper, the cefG gene with no intron is synthesized in vitro by Assembly PCR from 38 primers, which are complementary head and tail. The template of the gene is from the AJ404737 strain reported in NCBI. It was confirmed by DNA sequencing that there were nucleotide deletions at 3 sites in the synthesized cefG gene. The cefG is restored by site-directed mutagenesis method and verified by competitive PCR test. The restoration of each mutation site is through primers design, PCR amplification, Dpn I digestion, transformation, validation and sequencing, and at last the cefG gene matching the report of NCBI is got.In order to get the acetyltransferase with higher yield and purity for antibody preparation or Western blotting, the expression plasmid pET30-cefG was constructed in the following experiment, which a large number of recombinant proteins were expressed under 37℃. Most of the recombinant proteins are insoluble inclusion bodies. The recombinant bacteria is disrupted by ultrasonic, and then the insoluble part is washed with different-concentration urea. Finally the precipitation is dissolved with 8M urea, in which the purity of acetyltransferase is more than 96%, that is available to antibody preparation or Western blotting experiments.The purified inclusion bodies are vaccinated into 6-week old BALB/C female mice. About one month and a half later, tail blood is collected to obtain antiserum. The titer of antibody tested by enzyme-linked immunoassay (ELISA) is more than 8000, which shows that the antibody can meet requirement of the following experiments. First, calibration curve of the acetyltransferase concentration was determined by using the antibody. Second, the time curve of acetyltransferase during the fermentation of E. coli was drawn accordingly. These works prove the antibodies can detect acetyltransferase in vitro.Eukaryotic expression of plasmid pDH25-cefG is constructed with pDH25, which was presented by Professor D. Cullen in the University of Wisconsin as a gift, in order to recombine cefG into Acremonium chrysogenum genome for expression. Afterwards, protoplasts of Acremonium chrysogenum are prepared and the minimum inhibition concentration of hygromycin B is determined as 6μg·mL-1. At last the recombinant plasmids are tried to be transformed into Acremonium chrysogenum protoplasts by lectroporation'at different voltage.In this paper, rapid, efficient and accurate detection of acetyltransferase by ELISA was studied, and initial results about the work of genetically engineered microorganism for cephalosporin production is achieved. It is basis for further research on cloning and expression of cefG in cephalosporin producing microorganism.
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