A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase, equipped with a polyhistidine affinity tag, was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient purification of a recombinant luciferase with final yield 1.995mg/L of cell culture. Experiments on the recombinant luciferase also showed that the relative light units (RUL) of the enzyme were 5.8×108, and the specific activity was 2.9×1010 RLU/mg. By applying adenosine triphosphate (ATP) bioluminescence to detection of the coin bacteria using the recombinant protein, the ATP content of bacteria was 9.48×10-16mol/mL, and was identical to the bacteria counts (4500CFU/mL) in order of magnitude. Taken together, our results provided a simple and efficacious method of the preparation of recombinant luciferase, which could be applied in the determination of bacteria via ATP bioluminescence.

Objective:To explore the impact of cytochrome P450 oxidase (CYP2C19) *2 genotype on major adverse cardiovascular events (MACE) after percutaneous coronary intervention (PCI) and impact of MACE on blood stasis indexes .Methods :From Jan 2010 to Jan 2011 ,a total of 355 patients with blood stasis syndrome undergoing PCI were enrolled .All patients took aspirin and clopidogrel conventionally before PCI ,then maintained for one year at least after PCI .According to MACE or not ,patients were divided into MACE group (n=64) and no MACE group (n=291) .Turbidimetry method was used to measure platelet aggregation rate before and one week after PCI ,and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP ) was used to test CYP2C19 * 2 gene polymorphism .Results:(1) Compared with no MACE group ,there were significant rise in maximum platelet aggregation rate [MPA ,(36.38 ± 14.57)% vs .(44.87 ± 11.63)% ] and residue platelet aggregation rate [RPA , (25.76 ± 12.69)% vs .(34.45 ± 15.58)% ] , P< 0.01 both ;in percentage of CYP2C19*2 gene mutation (GA+AA) (36.43% vs .75.0% ,χ2 =27.832 ,P<0.01) in MACE group .(2) Multi-factor Logistic regression analysis in-dicated that >65 years ,CYP2C19*2 gene mutation (GA+AA) ,renal dysfunction (creatinine>1.5mg/dl) and re-duced left ventricular ejection fraction (LVEF< 50% ) were independent risk factors for MACE after PCI (OR=2.164~5.947 , P<0.05 or <0.01);(3) Binary linear correlation analysis indicated that CYP 2C19*2 gene muta-tion was positively correlated with RPA after PCI ( r=0.437 , P<0.001) .Conclusion:CYP2C19*2 gene mutation affects antiplatelet activity of clopidogrel ,so it′s helps to recognize MACE risk in patients with coronary heart dis-ease and blood stasis after PCI .

Objective To investigate the effect of neutrophil activation on pathogenesis of preeclampsia. Methods In 22 pregnant women with preeclampsia and 21 normal pregnant women at third trimester, neutrophil activation was examined by flow cytometry to assess the CD11b expression,and the levels of plasma endothelin 1(ET 1) and serum NO - 2 were also measured with no equilibrium radio immunoassay and with Griess assay respectively. Results The expression of neutrophil CD11b was significantly elevated in women with preeclampsia compared with that of normal pregnant women at third trimester [the mean fluorescence index of CD11b were 439 1? 169 1 and 321 2?166 3 respectively, P0 05). Conclusions The neutrophil CD11b expression is significantly elevated in preeclampsia. The increased neutrophil activation may cause the damage of vascular endothelium and result in compensatory increase of NO release in endothelial cells. It suggests that the neutrophil activation may play a key role in pathogenesis of preeclampsia.

Choroidal metastasis is one of the most common malignant tumors inside the eyes. It causes pain, hypopsia and some other related symptoms. It reduces the quality of the patients' life. It's significant for the patients to be detected and treated early, therefore they will have better vision and longer life. The treatments of choroidal metastasis are developing quickly. Both the vitreous cavity injection of targeted drug and gene therapy are hot topics of research. This paper summarizes the etiology, development, diagnosis and treatment of choroidal metastasis nowadays.

Objective Through To analyzing analyze the pathogen distribution and resistance analysis of elderly patients with pulmonary infection,in order to provide basis for clinical rational administration.Methods The clinical data of 285 elderly patients diagnosed as pulmonary infection were selected,and their sputum specimens cultivation were collected for statistical analysis,and the pathogenic bacteria distribution and drug resistance were analyzed.Results 292 strains of pathogenic bacteria were detected from 285 patients,of which 204 strains of gramnegative bacteria,accounting for 69.8％,mainly including pseudomonas aeruginosa,acinetobacter baumannii and klebsiella pneumoniae,while 84 strains of gram-positive bacteria,accounting for 28.8％,mainly including staphylococcus aureus and 4 strains of fungus,accounting for 1.4％.Different pathogens showed different degrees of antimicrobial resistance:pseudomonas aeruginosa,acinetobacter baumannii,klebsiella pneumoniae and e.coli had highly sensitivity for imipenem and meropenem,which were secondly sensitive to amikacin and highly resistant to ampicillin;Staphylococcus aureus and epidermis staphylococcus were all sensitive to linezolid and vancomycin,which were highly resistant to benzyl penicillin.Conclusion Gram-negative bacilli is the main bacteria for patients with pulmonary infection,which has higher antimicrobial drug resistant rate,and antimicrobial agents should be rationally used according to drug susceptibility results.

Objective To explore the correlation of serum HBV-DNA load,transaminase(ALT)and total bile acid (TBA)levels with hepatitis B virus (HBV)infection patterns to assess HBV infectivity.Methods The fluorescent quantitative PCR was used to detect serum HBV-DNA in 308 patients with HBV infection.ELISA was adopted to detect the HBV markers.The continuous mo-nitoring method was adopted to detect serum ALT and the cycling enzymatic method was adopted to detect serum TBA.Results The HBV-DNA content and serum ALT level had staitstical differences among the 3 infection patterns of HBsAb,HBeAg and HB-cAb positive,HBsAg,HBsAb,and HBcAb positive and 1,5 positive (P <0.05),the partial patients with HBeAg negative had the active HBV replication in vivo;HBV-DNA copy number in the patients with HBeAg negative was positively correlated with ALT level (P <0.05).Serum TBA level had no statistical difference among three knds of HBV infection patterns (P >0.05).Conclusion Serum HBV-DNA content and ALT activity have the correlation with the different HBV infection patterns,which could reflect the HBV replication and the infectivity intensity in some extent.

Objective:To explore the effect of different doses of lidocaine on recovery quality of patients with iodine-deficiency goiter after general anesthesia resection.Methods:Using a prospective design, 108 patients who underwent iodine-deficiency goiter resection in Yantai Yeda Hospital from April 2018 to April 2020 were selected as the research subjects. The patients were divided into control group (normal saline), lidocaine group A (1 mg/kg) and lidocaine group B (2 mg/kg) by random number table method, 36 cases in each group. The corresponding dose of lidocaine or the equivalent dose of normal saline was pumped into the vein after anesthesia induction and intubation for 30 minutes. The changes in the diastolic blood pressure (DBP), systolic blood pressure (SBP) and blood glucose (GLU) levels of the three groups were observed before administration, immediately after the operation, immediately after opening the eyes, immediately after extubation, and 10 minutes after extubation; at the same time, arousal and restlessness score (RS), sedation score (Ramsay), visual analogue score (VAS) and adverse reactions(coughing, nausea and vomiting, respiratory depression) occurrence were recorded.Results:At the moment of eye opening, immediately after extubation and 10 minutes after extubation, DBP (mmHg: 77.26 ± 4.85, 73.45 ± 4.37, 70.94 ± 4.45, 83.53 ± 6.19, 77.86 ± 5.33, 74.14 ± 5.20, 73.86 ± 5.73, 68.41 ± 5.16, 63.09 ± 5.24), SBP (mmHg: 129.17 ± 6.54, 121.25 ± 6.07, 116.24 ± 5.86, 137.48 ± 6.62, 129.53 ± 6.38, 123.07 ± 6.25, 125.65 ± 6.13, 117.48 ± 6.09, 108.17 ± 6.04), GLU level (mmol/L: 5.83 ± 0.36, 5.37 ± 0.29, 5.12 ± 0.26, 6.69 ± 0.34, 5.84 ± 0.30, 5.57 ± 0.27, 5.61 ± 0.32, 5.25 ± 0.31, 4.97 ± 0.28) of lidocaine group A and lidocaine group B were significantly lower than those of control group, the difference were statistically significant ( F=17.520, 25.755, 36.034, 41.028, 45.525, 74.330, 10.397, 49.756, 52.348, P < 0.05); among them, the DBP, SBP and GLU levels of lidocaine group A and lidocaine group B were lower than those of control group, and lidocaine group B was lower than lidocaine group A ( P < 0.05). The RS and VAS of lidocaine group A and lidocaine group B were significantly lower than those of control group, and Ramsay was higher than that of control group ( P < 0.05); and the RS and VAS of lidocaine group B were lower than those of lidocaine group A, the Ramsay was higher than that of lidocaine group A ( P < 0.05). The incidence of coughing, nausea and vomiting in lidocaine group A and lidocaine group B were lower than those in the control group ( P < 0.05); and there was no respiratory depression in the three groups. Conclusions:Thrity minutes after induction of anesthesia, continuous pumping of 2 mg/kg lidocaine can maintain blood pressure and GLU of patients undergoing general anesthesia for iodine-deficiency goiter, and effectively reduce restlessness, pain and adverse reactions during the recovery period. It is a satisfactory dose for thyroid surgery patients to wake up and comfortably.

Objective To investigate the effects of salvianolate on noradrenaline(NE), angiotension-Ⅱ(Ang-Ⅱ)and tumor necrosis factor-α(TNF-α)in rats with congestive heart failure. Methods Sixty male SD rats were randomly divided into 6 groups, the normal control group(NCG), the model group, the captopril group(CAG), the low dosage of salvianolate group(LSG), the high dosage of salvianolate group(HSG), the captopril and high doseage of salvianolate group(CSG). The rats of Congestive Heart Failure were established with peritoneal injection of adriamycin except the rats in normal control group. The rats in normal control group were injected with the equal volume of normal saline once per week for 6 weeks. The medication was started in every group at the same time. Eight weeks later, the Left Ventricular Systolic Pressure(LVSP), Left Ventricular End-diastolic Pressure(LVDP), +dp/dtmax, -dp/dtmax were measured. The serum levels of NE, Ang-Ⅱ and TNF-αwere measured. Protein expressions of Ang-Ⅱ and TNF-αin cardiac muscle were detected by Western blot. Results Compared with the model group[(90.77±14.75)mmHg, (22.52±2.58)mmHg, (3 290.16± 109.61)mmHg/s, (3 114.07±112.39)mmHg/s], HSG[(114.10±13.71)mmHg, (19.97±1.14)mmHg, (3 504.97 ± 163.94)mmHg/s, (3 303.02 ± 121.98)mmHg/s ] and CSG [ (141.18 ± 15.42)mmHg, (15.58 ± 1.46)mmHg, (3 766.56±159.93)mmHg/s, (3 566.70±154.57)mmHg/s]had significant difference in LVSP, LVDP, +dp/dtmax, -dp/dtmax(P<0.05 or 0.01). Compared with the model group[(85.06±9.07)ng/ml, (180.11±25.45)pg/ml, (205.80±15.73)pg/ml], the serum levels of NE, Ang-Ⅱand TNF-αwere decreased in groups of HSG[(75.33±8.60)ng/ml, (149.21±25.39)pg/ml, (188.84±13.79)pg/ml], CAG[(71.49± 6.21)ng/ml, (139.15 ± 24.83)pg/ml, (183.73 ± 10.99)pg/ml ] and CSG [ (60.02 ± 7.38)ng/m, (110.68 ± 28.63)pg/ml, (165.93±16.22)pg/ml]in different degree(P<0.05 or 0.01), those of CSG were significantly lower than CAG(P<0.05). Compared with the model group[(1.043±0.044), (1.167±0.048)], the expression of protein in Ang-Ⅱand TNF-α were decreased in groups of HSG[(0.981±0.024), (1.069±0.055)]CAG [(0.954±0.031), (1.046±0.053)]and CSG[(0.886±0.044), (0.955±0.038)]in different degree(P<0.05 or 0.01), those of CSG were significantly lower than CAG(P<0.05). Conclusion Salvianolate can reduce the serum levels of NE, Ang-Ⅱand TNF-αin rats with congestive heart failure, decrease the expression of Ang-Ⅱand TNF-αin its cardiac muscle, improve the cardiac function.

Glypican-3 （GPC3） is reported as a great promising tumor marker for hepatocellular carcinoma （HCC） diagnosis. Highly sensitive and accurate analysis of serum GPC3 （sGPC3）, in combination with or instead of traditional HCC marker alpha-fetoprotein （AFP）, is essential for early diagnosis of I-ICC. Biomaterial-functionalized magnetic particles have been utilized as solid supports with good biological compatibility for sensitive immunoassay. Here, the magnetic nanoparticles （MnPs） and magnetic microparticles （MmPs） with carboxyl groups were further modified with streptavidin, and applied for the development of chemiluminescence enzyme immunoassay （CLEIA）. After comparing between MnPs- and MmPs-based CLEIA, MnPs-based CLEIA was proved to be a better method with less assay time, greater sensitivity, better linearity and longer chemiluminescence platform. MnPs-based CLEIA was applied for detection of sGPC3 in normal liver, hepatocirrhosis, secondary liver cancer and HCC serum samples. The results indicated that sGPC3 was effective in diagnosis of HCC with high performance.

A chemiluminescence enzyme immunoassay based on magnetic microparicles (MmPsCLEIA) was developed to evaluate serum α-fetoprotein (AFP) in parallel with tramional colorimetric enzyme-linked immunsorbrnt assay (ELISA).A sestematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer of immunoreagents,less total assay time,and better linearity,recovery,precision,senitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results werecompared with commercial electrochemilunminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R2=0.6703; however,the correlation between MPs-CLEIA and ECLIA (R2=0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R2=0.6866).