The Clone And Expression Of PBP 2x And Detection Of Penicillins Residue In Milk

With the rapid development of dairy industry, China's quality supervision of dairy products has become increasingly strict. In 2007, China adopted the "Raw Dairy Standards (Amendment ) ", which listed antibiotics residue test as mandatory testing items. At present, many methods concerning antibiotic residues in milk are reported, such as microbial growth inhibitor methods, physical and chemical analysis and immune analysis. However, the above methods are either time-consuming, operating-complex, or costly, which failed to meet the need of the dairy industry. Therefore, a more accurate, simple, and time-saving testing method is required to develop.Since PBP 2x has strong affinity forβ-lactam antibiotics to the nature, the paper proposes a novel microplate assay for the determination of penicillins with intact beta-lactam structure in milk. Researches in this paper have been done as follows:1 Penicillin-binding protein PBP 2x from Streptococcus pneumoniae is selected as target protein; primers are designed according to its genetic sequences.2 The amplified target gene is connected with the cloning vector to generate pGEX -PBP 2x by using PCR technology, and then pGEX -PBP 2x is transformed into E.coli BL21.3 Taking GST activity and the concentration as indicators, this paper makes Single-factor test on the bacterial ultrasonic broken time and the main factors that influent fusion protein GST-PBP 2x in E. coli expression, including E. coli strain, transformation of bacteria, IPTG final concentration and induction time. It designs rotation orthogonal combination test based on induced temperature and time, from which an experience model and the optimized value of induced temperature and time by genetic algorithm are obtained.Reorganization of BL21 expressed in GST-PBP 2x is induced under parameters in the best conditions. Cell broken up by ultrasound is centrifugalized and then the supernatant is electrophoresed. Through software analysis, recombinant protein GST-PBP 2x takes about 15 per cent of total cell protein, (GST at this time measured the activity of 21.26/min mL) which achieves a high expression of GST-PBP 2x.4 GST affinity chromatography technology is used to get isolated GST-PBP2x, and then use thrombin to digest GST-PBP 2x; to make digested solution transflux Sepharose 4B affinity chromatography columns to remove GST protein; make the solution transflux S-100 gel chromatography to remove thrombin and get concentrated PBP2x protein; after the SDS-PAGE gel imaging systems analysis, its purity is over 99% that can meet the needs of applied research.5 Bradford method is applied to detect the rate of protein residues in PBP 2x supernatant solution under different pH conditions. The lowest rate of pH 5.0 in PBP 2x supernatant solution is the isoelectric point of protein.6 Molecular docking between PBP2x and four commonly usedβ-lactam antibiotics in veterinary clinic is simulated to generate their total energy and sort them according to their affinity.7 A dynamic model is established about PBP 2x andβ-lactam antibiotics, and taking ampicillin for example, through the measurement of the marked test, the combination of curves and dissociation curve are got, and parameters of dynamic model about ampicillin and PBP2x are: k₁=9.21×10⁴ L/mol.s k₂= 4.81×10^-4 s^-1 K_d=5.22 nmol/LIt is ultimately proved that PBP 2x andβ-lactam antibiotics have high affinity, which is sufficient to meet the rapid determination ofβ-lactam antibiotics residues in milk.8 The penicillin-binding protein PBP 2x from Streptococcus pneumoniae has been utilised to develop a novel microplate assay for the determination of penicillins with intact beta-lactam structure in milk. In the assay, the receptor protein is immobilised to a microplate in the first step. To each sample a bifunctional reagent is added, with ampicillin and biotin as functional groups (B-AMPI). The amount of bifunctional reagent, which is bound via its ampicillin part to the receptor protein, decreases with increasing beta-lactam concentration in the sample. The detection step uses avidin Fab fragments marked with horseradish peroxidase. The more bifunctional reagent is bound to the receptor protein, the more avidin fragments are bound via the biotin part of the reagent. A maximum colour development with tetramethylbenzidine as chromogen for the peroxidase reaction is achieved, when no beta-lactam residues are present.The assay has been developed as screening test with the option for a quantitative assay, when the identity of the residue beta-lactam is known (e.g. Penicillin G in this paper). Penicillin G could be detected at levels corresponding to 1/2 EU maximum residue limit (MRL) in milk without needing lengthy and elaborate sample pre-treatment. Matrix calibration curves are presented, which show that quantitative analyses are possible. 9 The penicillin-binding protein PBP 2x from Streptococcus pneumoniae has been utilised to develop a novel microplate assay for the determination of ceftiofur with intact beta-lactam structure in milk. The assay has been developed as screening test with the option for a quantitative assay for ceftiofur residues. Ceftiofur could be detected at levels corresponding to 1/5 EU maximum residue limit(MRL) in milk without lengthy and elaborate sample pre-treatment. Matrix calibration curves are presented, which show that quantitative analyses are possible.In short, this paper successfully realizes the cloning and expression of PBP2x and optimizes the culture conditions of E.coli BL21 in order to maximize the expression of PBP 2x. Secondly, this paper simulates Molecular docking between PBP 2x andβ-lactam antibiotics and calculates the dynamic parameters by experiments. Finally, it applies PBP2x into the determination ofβ-lactam antibiotics residue in milk and proposes two new detection methods:A Receptors analysis detect penicillin G residue in milk by indirect competitionB Receptor and ELISA analysis detect directly ceftiofur residues in milkThe results show that this method has high sensitivity, a high degree of automaton, and a good stability and reproducibility. It is easy to operate and cheaper. It can be applied to dairy industry as a rapid screening method.