Optimized inverse PCR strategy for constructing multilocus mutants efficiently / 生物工程学报

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.

Analysis of the Safety of Antibiotic Drug Use by Children Being Referred and Hospitalized under Medical Consortium Model / 中国药房

OBJECTIVE:To provide reference for safe application of antibiotic drugs in single or inter-medical institution ser-vices. METHODS:An antibiotic drug safety evaluation index system was established by reference to literatures. Medical records tracing method was adopted to obtain the medical records of children with respiratory infectious diseases under the medical consor-tium model. The medical records were matched and evaluated by experts to analysis the safety of antibiotic drug use in single medi-cal institution and the connection between two medical institutions with respect to the safety of antibiotic drug use. RESULTS:248 effective medical records had been obtained. Under the medical consortium model,the rate of the combination of antibiotic drugs in large hospitals was 86.29%,higher than the community hospitals of 77.02%(χ2=5.49,P<0.05). The error rate of antibiotic drug use by children in large hospitals was 14.52%,lower than the community hospitals of 25.81%(χ2=9.733,P<0.05). There were many contradictions in antibiotic drug use between the medical institution where children received treatment initially and the medi-cal institution which the children were referred to and hospitalized in. There were totally 128 cases of unsafe antibiotic drug use, with the overall incidence of 51.61%. CONCLUSIONS:Under the medical consortium model,the safety of antibiotic drug use by the hospitalized children in single medical institution is worrying,and the connection between two medical institutions with respect to the safety of antibiotic drug use by children who are referred and hospitalized is less reliable.