目的 研究从大肠埃希菌宿主提取基因工程RNA同时去除内毒素的条件.方法 将串联的小鼠短散布核元件(short interspersed nuclear elements,SINE)B1插入pET-28a(pET)构建pET-B1×16反序质粒(pET-B1 as),转化大肠埃希菌BL21(DE3),获得pET-B1 as-DE3菌.用TRlzol法、SDS-热酚法、SDS-NaCl离心法和SDS-NaCl过滤法从pET-B1as-DE3菌制备基因工程RNA,Triton X-114相分离法进一步去除内毒素,检测制备的RNA中内毒素含量及RNA产量、纯度、完整性,并进行小鼠体内毒性试验和家兔热源试验.结果 TRlzol法制备的RNA产量低;SDS-热酚法制备的RNA中内毒素含量高;SDS-NaCl过滤法去除内毒素的效果优于SDS-NaCl离心法,同时具有较高的RNA产量及较好的RNA完整性.小鼠静脉注射用SDS-热酚法和SDS-NaCl过滤法制备的RNA,前者引起小鼠不良反应,后者未引起小鼠不良反应.用Triton X-114相分离能进一步去除由SDS-NaCl过滤法制备的RNA中的内毒素,家兔热源试验证明SDS-NaCl过滤法联合Triton X-114相分离法制备的RNA,其热原符合动物体内试验要求.结论 建立了用SDS-NaCl过滤法联合Triton X-114相分离法制备基因工程RNA的方法,可有效去除RNA中污染的内毒素.

目的 建立快速检测实验大鼠冠状病毒和仙台病毒的双重PCR方法.方法 根据大鼠冠状病毒N基因、仙台病毒L基因设计特异性引物;经过双重PCR优化,特异性和敏感性的检测,建立双重PCR体系.应用该PCR体系检测人工感染仙台病毒组织DNA样本和实验动物组织样本,并与ELISA方法比对.结果 双重PCR扩增出大鼠冠状病毒(168 bp)和仙台病毒(262 bp)目的条带,PCR扩增产物测序结果利用核酸BLAST功能进行同源序列对比,仙台病毒和大鼠冠状病毒同源性分别为100％和99％.仙台病毒和大鼠冠状病毒的检测下限为1.56×102 copies/μL.特异性检测对小鼠肝炎病毒扩增,产生片段大小近似大鼠冠状病毒产物.应用建立的双重PCR体系检测人工感染仙台病毒组织DNA样本,30份DNA标本均被检出;检测94份实验动物肺组织样本,结果均阴性.结论 建立的双重PCR方法操作简单、快速、特异性强、灵敏度高,能够实现对实验动物仙台病毒和大鼠冠状病毒病原体的快速检测.

Objective Aiming at detecting Staphylococcus aureus、Pseudomonas aeruginosa and Klebsiella pneumoniae in laboratory animals,the paper provides a rapid,sensitive and simple test method.Methods According to Staphylococcus aureus nuc gene,Pseudomonas aeruginosa LasI gene,Klebsiella pneumonia PhoE gene and general 16S rRNA gene, designed specific primers;Through the optimization of multiplex PCR primer concentrations and annealing temperature, the specificity and sensitivity of detection, establishing multiplex PCR system.Application of the PCR system test specimens of artificial infections and experiment animal feces is compared with traditional test method.Results Multiplex PCR amplification of Staphylococcus aureus (153 bp), Pseudomonas aeruginosa (600 bp) with Klebsiella pneumoniae (368 bp) and general (520 bp).The multiplex sensitivity for the purpose of 10pg, specificity of detection was not detected from other pathogens.Application of establishing multiplex PCR system to detect the artificial positive samples, and detect 1 Pseudomonas aeruginosa positive case in 76 fecals.Conclusions This paper established the multiplex PCR method which has the advantages of specific,sensitive,simple and rapid, and provides a reliable way for rapid test in laboratory animals microbiology.

Objective To establish and compare atherosclerosis models of Wistar closed colony rats and high fecundity inbred HFJ rats by feeding high-fat diets and immune injury. Methods Male Wistar rats and HFJ inbred rats were randomly divided into normal group and model group. The normal group was fed with basic diet. The model group was fed a high-fat diet and received immune injury ( bovine serum albumin 40 mg / kg,chicken egg albumin 2. 5 mg / kg) and gastric perfusion of vitamin D3. The serum lipid level,biochemical indices and pathological changes were examined after 90 days. The expression of vascular endothelial growth factor ( VEGF) in the aortic arch was detected by immunehistochemical tech-niques. Results ( 1) In normal group,the values of TG,TC and LDL-C in HFJ inbred rats were significantly higher than in Wistar rats ( P 0. 05) . The production of LDL-C in model group of HFJ rats also showed a higher level than in Wistar rats ( P 0. 05) ; ( 2) The content of CK and CK-Mb in model groups were statistically higher than those of normal animals ( P 0. 05) ; ( 3) After HE staining,typical pathologic stage Ⅲ characters of atheromatous plaques was observed in the model group of HFJ inbred rats rather than in Wistar rats; ( 4) The expression of VEGF in the model group HFJ inbred rats was higher than in Wistar rats as determined by immunehistochemical techniques ( P 0. 05) . Conclusion An atheroscle-rosis mouse model was successfully developed based on HFJ inbred rats,which could provided a new strain of laboratory animal for AS researches.