Objective To construct an expression vector pGEX-2T/N, and to express the fusion protein consisting of N protein of SARS-CoV in E. coli.Methods The N region gene of SARS-CoV was obtained by RT-PCR. The expression vector PGEX-2T/N was constructed by DNA recombination. The recombinant plasmid was transformed into E. coli BL21(DE3). The expression of the fusion protein was determined by Western blot with anti-SARS-CoV antibody positive blood sera. Results The N region gene of SARS-CoV was obtained. The fusion protein GST-N was soluble. Western blot analysis showed that the reaction of GST-N to anti-SARS-CoV sera was positive. Conclusion The pGEX-2T/N has been constructed and expressed in the form of fusion protein GST-N successfully, and the result lays the foundation for further study of SARS-CoV N protein.

Objective To prepare and characterize the polyclonal antibody against N protein of SARS-CoV. Methods The polyclonal antibody against SARS-CoV N was obtained from immunized rabbit with purified GST-N. The titer of the antibody was determined by indirect ELISA, and the specificity by Western blot and immunochemical staining. Results The rabbit′s antibody against SARS-CoV N was prepared successfully. The titer of antiserum against SARS-CoV N was about 1.2?10~ -5 . Western blot and immunochemical staining analysis showed that the polyclonal antibody could bind to the expressed fusion protein specifically. Conclusion The rabbit′s antibody against SARS-CoV N has been prepared successfully, and it can be a useful reagent for clinical diagnosis and further research.

Objective To compare the cellular immune responses induced by two different vectors,eukaryotic plasmid pcDN3.1(+) and attenuated typhia live vaccine Ty21a,-associated mimic epitopes of HCV hypervariable region 1,so to find a better way for vaccine immunization.Methods The DNA sequence based on the peptides' sequence of HCV hypervariable region 1 related consensus polymimotopes was synthesize and then inserted into plasmid pcDN3.1(+) to construct recombinant plasmid pcDN3.1(+)-SP,which then was transfected into live attenuated typhia vaccine Ty21a to construct Ty21a-SP.The mice were immunized orally with Ty21a-SP and intramuscularly with pcDN3.1(+)-SP,respectively,and then sacrificed by exsanguination.The splenocytes were separated and restimulated with pooled synthesized peptides,and then collected.Flow cytometry was employed to identify CD8+IFN-?+ T cells,and non-radioactive MTS method was adopted to test T cell proliferation,and non-radioactive LDH method was used to test cytotoxic T cytolytic reaction(CTL).Results Compared with control groups,the proliferation of splenocytes was apparently enhanced,the proportion of CD8+IFN-?+ cells obviously increased,and CTL responses also significantly increased after the spenocytes of mice immunized by pcDN3.1-SP and Ty21a-SP were restimulated with synthesized peptides.And the responses mentioned above in the mice immunized by Ty21a-SP were stronger than those mice immunized by pcDN3.1-SP.Conclusion Using attenuated typhia live vaccine Ty21a as DNA vector is an effective way to induce cellular immune responses.

Objective:To analyze the factors affecting lymph node failure pattern after radiotherapy/chemotherapy in esophageal squamous cell carcinoma without initial clinical metastasis. Methods:A total of 263 patients, who were diagnosed as thoracic esopha-geal carcinoma from January 2002 to December 2009, were included in this retrospective study. Factors affecting lymph node failure pattern with general clinical data and tumor local factors were analyzed. Results:Among the 263 esophageal cancer cases, 31 (11.8%) had lymph node metastasis after treatment, including 18 cases of simple lymph node metastasis and 13 other cases of lymph node metas-tasis with esophageal and other organ metastasis or recurrence. The numbers of cases for lymph node metastasis in the upper, middle, and lower thoracic esophagus were 11 (13.3%), 13 (10.1%), and 7 (13.7%), respectively. Univariate analysis showed that recent cura-tive effect, length of tumor on X-rays, maximum tumor diameter, and tumor volume were the significant factors associated with lymph node metastasis (χ2=7.597, 9.717, 5.361, and 4.815;P=0.006, 0.002, 0.021, and 0.028). Logistic regression analysis results showed that recent curative effect and length of tumor on X-rays were independent significant factors (P=0.004 and 0.026). Conclusion:Recent cu-rative effect and length of tumor on X-rays were the significant factors associated with lymph node failure pattern after radiotherapy/chemotherapy in esophageal squamous cell carcinoma without initial clinical metastasis.

Objective To explore the in vitro cytotoxicity of sunitinib in highly metastatic hepatocellular carcinoma cell line MHCC97-H, and the effect of it on the expression level of focal adhesion kinase (FAK). Methods MHCC97-H hepatoma cells were cultured and divided into control group and experimental (sunitinib) group. Experimental groups were added 2.5, 5,10 and 20μmol/L of sunitinib for 24, 48 and 72 hours respectively. The morphological changes were observed before and after sunitinib treatment in MHCC97-H with Giemsa stain. The inhibitory rate of proliferation in MHCC97-H was detected by MTT assay. The expressions of FAK protein before and after sunitinib treatment were detected by Western blot assay. Results Sunitinib showed the inhibitory effect on hepatoma cell line MHCC97-H. Giemsa staining showed that chromatin condensation, nuclear fragmentation, apoptotic bodies and other typical morphological features. The inhibitory rate was the most obvious in 48-h treatment group. The inhibitory rates were (0.433 ± 0.115)%, (32.863 ± 1.471)%, (49.240 ± 2.256)%, (63.797±2.707)%and (58.887±3.409)%for 2.5, 5, 10 and 20μmol/L concentration groups, and there were signifi-cant differences between groups (P<0.05). Results of Western blot assay showed that the expression levels of FAK protein were significantly reduced after different concentrations of sunitinib treatment for 48 h (P<0.05). Conclusion Sunitinib has inhibitory effect on hepatoma cell line MHCC97-H, enhances the apoptosis and decreases the the expression of FAK pro-tein.

Objective To evaluate the value of diffusion weighted imaging (DWI)in distinguishing the solid solitary pulmonary nodule (SPN).Methods 42 patients with SPN (malignant in 25 and benign in 1 7)who were confirmed by operation,biopsy or follow up after treatment underwent routine chest T1 WI,T2 WI and DWI.The b values were chosen as 300,500,800 and 1 000 s/mm2 ,and the corresponding apparent diffusion coefficient (ADC)values and the signal intensity (SI)were respectively measured.Results The ADC values and SI of benign and malignant SPNs were gradually reduced with increasing b value.The ADC value between benign and malignant SPNs was statistically significant with b value of 500 s/mm2 (P 500 =0.03 <0.05 ),meanwhile the SI was statistically significant with b values from 300 to 1000 s/mm2 (P 300 <0.001,P 500 =0.03 <0.05,P 800 =0.01 <0.05, P 1 000 =0.02<0.05).Conclusion Both SI and ADC value of DWI play important role in distinguishing benign and malignant SPNs, and the diagnostic efficiency of SI is superior to ADC value.

To design a mobile device for infants appeasing in order to improve CT of the infant. The device was composed of the components of broadcasting, supporting and monitoring. The broadcasting component consist-ed of a high-definition 14-inch monitor and a DVD player, the supporting component was made up of the bar connector, base supporter and fastening bolt, and the monitoring component included a camera and a monitor. The head CT exami-nation results with the device were compared with those without the device. The device could significant improve the head CT examination of the infants. The device can enhance infants CT examination by appeasing them, and thus is worthy popularizing practically.

Objective To analyze the correlation between intestinal flora changes and neonatal necrotizing enterocolitis (NEC)through 16S rRNA metagenomic sequencing and bacterial culture. Methods From September 2018 to March 2019, 10 NEC cases and 6 controls were randomly selected in the neonatal ICU ward of Nanjing maternal and child health care hospital to analyze the 16S rRNA metagenomic diversity of the for intestinal flora. The fecal samples and corresponding environmental samples were corrected from 51 cases of NEC children and their case controls to isolate and culture Clostridium. Results The dispersion of samples within the case group was smaller than that of the control group, and the sample diversity was higher than that of the control group. In the isolation and culture of Clostridium, the overall detection rate of Clostridium in the case group was 43.14% (22/51), and the detection rate of Clostridium butyricum was the highest (19.61%, 10/51). There was a statistical difference between the two groups (χ²=5.85, P=0.015 58). All Clostridium strains did not carry the A, B and E type neurotoxin genes. Conclusion: Increased intestinal flora diversity, intestinal flora abundance and changes in the abundance of Clostridium may be closely related to the intestinal environment of children with NEC; Clostridium, especially Clostridium butyricum, may be related to the occurrence of NEC.