This paper reports the results of a study about the segregational stability of a series of pNK289-derived plasmids. The segregational stability in B. subtilis AS1. 1176 was found to be different among these same originated plasmids. The difference in segregational stability was shown not to be related to the sizes or replication patterns of the plasmids, but to have some relation to their copy numbers. As the unstable plasmid pNK219 can be miaintained stably in B. subtilis BD 224, it can be inferred that the host inheritable background could affect the segregational stability of plasmid. These results not only have laid a foundation for finding out some genes related to the stability of pNK289-derived plasmids, but also provided theoretical basis for constructing stable recombinant plasmid in Bacillus.

Objective: To express the protein of HPV18E6 based on pET-32a(+) at high level and study the expression and significance of HPV18E6 proteins in laryngeal carcinoma. Methods: The HPV18E6 gene was amplified by PCR and cloned into pET-32a(+). The amplified fragment was inserted into the plasmid pET32a (+) that was digested with BamHⅠand Hind Ⅲ. The recombinant plasmid pET32/E6 was transformed into E.coli JM109 which was selected with ampicillin. The recombinant plasmids were successfully introduced into E.coli BL21(DE 3) and were induced by IPTG. SDS-PAGE and Western blot analysis were used to detect the confusion protein. Finally, the optimization of expression conditions, such as temperature, concentration of IPTG, was studied. Results: The recombinant plasmids were identified and confirmed with enzyme digestion and sequencing. The BL21(DE3) transformed recombinant plasmid pET32/E6 had expressed HPV18E6 recombinant protein effectively. The optimum conditions of expression were 37 ℃, 1 mmol/L IPTG. Conclusion:Prokaryotic expression vector pET-32a(+)-HPV18E6 was successfully constructed. The high-level expression of HPV18E6 was achieved in E.coli BL21(DE3).

This study was aimed to observe the effects of acupuncture combination with medicated thread moxibustion ofZhuang nationality medicine on neurotransmitters in rats with sleep deprivation. A total of 80 SPF grade healthy Wistar rats were randomly divided into the control group, model group, treatment group and diazepam group with 20 rats in each group. The establishment of insomnia model was through sleep deprivation in the model group, treatment group and diazepam group. Based on the treatment of the model group, the treatment group was treated by acupuncture combination with medicated thread moxibustion ofZhuang nationality medicine, once a day for continuous 14 days. Intragastric administration of diazepam according to 3 mg·kg-1 body weight was given in the diazepam group. While intragastric administration of the same volume normal saline was given to the control group once a day for continuous 14 days. And then, the levels of orexin, NE, 5-HT, CRH and ACTH were detected. The expressions of orexin mRNA and c-Fos were observed. The results showed that compared with the control group, the levels of orexin, ACTH, 5-HT, NE and CRH increased or obviously increased in the model group with significant difference (P < 0.05, orP< 0.01). The expressions of orexin mRNA and c-Fos in the model group were increased or obviously increased with significant difference (P < 0.05, orP< 0.01). Compared with the model group, the levels of orexin, ACTH, 5-HT, NE and CRH reduced or obviously reduced with significant difference in the treatment group (P < 0.05, orP < 0.01). The expressions of orexin mRNA and c-Fos were reduced or obviously reduced with significant difference in the treatment group (P <0.05, orP < 0.01). It was concluded that acupuncture combination with medicated thread moxibustion ofZhuang nationality medicine took an effect on the treatment of insomnia by mediating neurotransmitters associated with hypothalamic-pituitary-adrenal axis (HPA axis) through orexin, which was probably related with its therapeutic mechanism.

Prokaryotic expression vector of mouse HPV16E6 gene was constructed. A pair of primers were designed according to the digestion sites in plasmid pGEX-KG and the HPV16E6 gene sequence published by GenBank. The DNA fragment of 321bp was amplified by PCR from the HPV recombinant plasmid with HPV16E6 gene, then cloned into pGEX-KG and transformed into the host E. coli strain JM109. The fragment was conformed to the original sequence, which indicated that fusion expression vector pGEX-KG-HPV16E6 was constructed. The pGEX-KG-HPV16E6 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degrees C, the expression product of HPV16E6 gene was identified by SDS-PAGE and Western blot. HPV16E6 fusion protein had been expressed successfully in the form of inclusion bodies, the molecular weight of fusion protein being 38 kD. Meanwhile, the optimum condition of HPV16E6 fusion protein expression was induced with 1.0 mmol/L IPTG for 4h. The fusion protein reacted specifically with the antibodies against HPV16E6. HPV16E6 gene was successfully expressed in E. coli, which could be used as a basis for preparing HPV16E6 vaccine in human.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Repressor Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

Objective To evaluate the clinical effect of milrinone nebulized inhalation for improving intraoperative cardiac function and pulmonary arterial pressure in the patients with chronic obstructive pulmonary disease (COPD) complicating pulmonary hypertension (PH).Methods Forty-four surgical patients with COPD complicating PH in the Chongqing Municipal Medical Emergency Center from June 2015 to June 2016 were chosen,including 23 cases of thoracic surgery,13 cases of abdominal surgery and 8 cases of lower extremity fracture surgery.The patients were divided into the control group and treatment group,22 cases in each group.The control group received the routine comprehensive treatment.In addition receiving the conventional comprehensive treatment,milrinone nebulized inhalation in the treatment group was given before general anesthesia.Both of the two groups were imbedded with floating catheter in the right internal jugular vein for detecting the clinical indexes:cardiac output (CO),pulmonary artery systolic pressure (PASP),pulmonary artery mean pressure (PAMP) and pulmonary capillary wedge pressure (PCWP).Results Compared with before treatment,CO after treatment in the treatment group was significantly increased (P＜0.05),PASP,PAMP and PCWP after treatment in the treatment group were significantly decreased(P＜0.05).CO,PASP,PAMP and PCWP in the control group had no statistical difference between before and after treatment,the difference was not statistically significant (P＞0.05).Conclusion Intraoperative milrinone nebulized inhalation in the patients with COPD complicating PH can effectively improve the patient's cardiopulmonary function.

OBJECTIVE To explore standardized evaluation process for clinical comprehensive evaluation of blood lipid- regulating drugs and perform rapid assessment of clinical comprehensive evaluation of blood lipid-regulating drugs with different mechanisms so as to provide reference for the drug catalogue selection and rational drug use of medical institutions. METHODS Referring to guidelines and consensus such as the guideline for the management of comprehensive clinical evaluation of drugs， the methods such as literature research， expert interviews， and Delphi expert consultation were used to establish a multi-dimensional and multi-criteria clinical comprehensive evaluation index system and quantitative scoring table for blood lipid-regulating drugs around the two main lines of technical evaluation and policy evaluation. Then 13 blood lipid-regulating drugs with different mechanisms in 21 third-grade class-A medical institutions from five provinces and regions of Northwest China were scored from both technical and policy dimensions to form a comprehensive evaluation result. RESULTS The clinical comprehensive evaluation index system and corresponding rapid evaluation quantitative scoring table were constructed for blood lipid-regulating drugs in the five northwest provinces and regions. The technicalevaluation section included 6 primary indicators， 13 secondary indicators， and 34 tertiary indicators， totaling 110 points. The policy evaluation section included 4 primary indicators and 6 secondary indicators， with a total score of 40 points （30 points for some drugs） and a total score of 150 points （or 140 points）. The scoring results showed that the highest score was atorvastatin， followed by rosuvastatin and simvastatin. CONCLUSIONS Statins are still the cornerstone of drug therapy for patients with dyslipidemia； the rapid evaluation quantitative scoring table constructed in this study is comprehensive， systematic and operable. The evaluation process in this study can provide empirical references for other groups to exploring the standardized path and quality control mechanism of clinical comprehensive evaluation of drugs.