Objective To perform the amplification ,sequencing and prokaryotic expression of APH (3′′)‐Ⅰ and AAC (2′)‐Ⅰgenes from the clinically isolated gzch810 strain(SM gzch810)of Stenotrophomonas maltophilia to provide the basic materials for the next step functional test .Methods The SM gzch810 genome chromosome was extracted ,the APH (3′′)‐Ⅰ ,AAC (2′)‐Ⅰ whole genes were amplified by PCR and sequenced after being cloned into pMD18‐T vector .The recombination were subcloned into pGEX‐4T‐1 vector and the expression of the recombinant APH (3′′)‐Ⅰ and AAC (2′)‐Ⅰ were analyzed by SDS‐PAGE .Results The 800bp and 550bp DNA fragments of APH(3′′)‐Ⅰ ,AAC(2′)‐Ⅰ gene were amplified from SM gzch810 by PCR and sequenced ;the sequence comparison analysis showed that DNA and amino acid sequence identities of APH (3′′)‐Ⅰand AAC (2′)‐Ⅰ genes with other strains were 91% and 95% respectively .The sequence of APH (3′′)‐Ⅰand AAC(2′)‐Ⅰ of SM gzch810 were submitted to GenBank(accession number :HQ315852 and HQ315853);two major protein bands corresponding to the expected recombinant GST‐TP fusion proteins (56 × 103 and 46 × 103 respectively) were identified by SDS‐PAGE .Conclusion APH(3′′)‐Ⅰand AAC(2′)‐Ⅰgene of SM gzch810 are successfully cloned and expressed ,which lays a good foundation for further detecting corresponding antibi‐otic resistance and functional evaluation of above two kinds of recombinant E .coli .

Objective To conduct the amplification,cloning,bioinformatics analysis,prokaryotic expression and purification of enterovirus 71 VP1 gene segment and to initially confirm the biological activity of the recombinant expression product.Methods A pair of specific primers was designed according to GenBank EV71 sequence,viral RNA as a template was extracted from the throat swab specimens in the EV71 patients.EV71 VP1 gene was amplified by RT-PCR.After enzyme digestion,the expression vector pET28a was inserted.The prokaryotic expression vector of pET28a-EV71 VP1 was constructed.Then the E.coli DH5a transforma-tion was performed.IPTG was adopted for induction expression.The expression results were analyzed by using SDS-PAGE and Western blot.The bioinformatics analysis of the sequenced results was performed by the software.Expressed protein was purified and the plates were coated,ELISA was used to test the VP1 specific IgG antibody in serum samples of EV71 positive and COX A16-positive patients.Results The BLAST alignment showed that the homology of the objective gene EV71 VP1 was 99% com-pared with other strains(JQ766207.1)in GenBank.EV71 VP1 protein was about 32×103 ,which mainly existed in the form of in-clusion body.The bioinformatics analysis showed that EV71 VP1 protein was a hydrophilic protein,without transmembrane region and N-terminal signal peptide sequence,the tertiary structure existed.The ELISA results showed that the specific IgG OD value in EV71-positive patients was(2.425±0.521),OD value in COX A16 positive patients was(1.205 ±0.314),the normal control OD value was(0.353±0.128).The sensitivity and specificity of EV71 VP1 protein detection were 84% and 88% respectively.Conclu-sion The pET28a-EV71 VP1 expression vector is successfully constructed;the preliminary analysis on the serum of the infected patients by ELISA shows that the obtained objective protein has higher sensitivity and specificity,which is initially confirmed to have biological activity and can be further used for the related study on EV71 diagnosis and vaccine.

Objective:To study the feasibility of transplantation of normal rat penile corpus cavernosum and major pelvic ganglion (MPG)into the renal subserous region of a Nu /Nu mouse based on allograft technology.Methods:Penile corpus cavernosum and MPG,harvested from Sprague-Dawley (SD)rats under sterile condition,were transplanted underneath the kidney capsule of Nu /Nu mice through the mi-crosurgery instruments and surgery microscope.The histopathologic changes and cellular proliferation in the transplanted penile corpus cavernosum and MPG were then analyzed at the end of 1week and 4 weeks after transplantation.Histological staining and immunohistochemical staining were used to evaluate the main outcome measures.Results:After 1 week,the tissue morphology of the transplanted corpus caverno-sum underneath the kidney capsule of Nu /Nu mice was consistent with normal penile corpus cavernosum, and blood could be observed in the penis cavernous sinus of the graft;after 4 weeks,the mophorlogy of the tranplanted corpus cavernosum near the kidney was consistent with normal penile corpus cavernosum, while fibrosis was noteworthy in the graft away from the kidney,but blood could still be seen in the penis cavernous sinus.After 1 week,the tissue morphology of the transplanted MPG was consistent with normal MPG,multiple islet-like cell clusters could be seen in the transplanted MPG in the renal subserous re-gion,and angiogenesis could be observed near the kidney;after 4 weeks,a network of blood vessels was clearly visible away from the kidney,and islet-like cell clusters were still clearly observed in the trans-planted MPG.In addition,ki67 positive cells were observed in the transplanted penile corpus cavernosum and MPG after 4 weeks of transplantation,which indicated that there was still cell proliferation activity in the grafts.Conclusion:The transplanted corpus cavernosum and MPG underneath the kidney capsule of Nu /Nu mice could survive at least 4 weeks.Moreover,the inner structure of the transplanted corpus ca-vernosum and MPG was close to the normal tissue.The underlining mechanism may be related to the lo-cal microenvironment underneath the kidney capsule of Nu /Nu mice and the neovascularization in the transplanted grafts.

Objective To investigate the effect of Salvianolic acid on endoplasmic reticulum stress (ERS) pathway in brain hippocampus of PAP mice. Methods Twenty PAP dual transgenic male mice were selected, they were randomly divided into a PAP mice model group and a Salvianolic acid group, 10 mice in each group; another 10 SPF grade C57BL/6J male mice were selected as a normal control group. In the Salvianolic acid group, 0.9% normal saline solution of Salvianolic lyophilized injection (400 g/L) of dosage 21 mg·kg-1·d-1 was injected intravenously through a tail vein of mice; the PAP mice model and normal control groups were given the same amount of 0.9% normal saline, and the therapeutic course was consecutive 4 weeks in the three groups. At the end of the 4th week, the Morris water maze test was carried out to observe the changes of escape latency, the third quadrant residence time (RTQ), entry angle into water and cross-platform times of mice in each group; amyloid precursor protein (APP) positive cell expression in cerebral hippocampus of mice were detected by immunohistochemistry; Western Blot was used to detect the expression level of PER like endoplasmic reticulum kinase-eukaryon initiation factor 2α-C/EBP homogenous protein (PERK-eIF2α-CHOP) pathway related proteins in hippocampus of mice. Results The escape latency of the PAP mice model group on the 1st to 5th day were significantly longer than those of the normal control group, although a downward trend was observed on the 5th day, it was still significantly longer than that of the model group (seconds: 58.41±2.36 vs. 28.60±10.15); compared with the PAP mice model group, the escape latency of Salvianolic acid group was shorter at each time point, and reached the shortest level on the 5th day (seconds: 31.97±8.36 vs. 58.41±2.36). In the PAP mice model group, the RTQ and the number of crossing platforms were significantly lower than those in the normal control group [RTQ (seconds): 8.27±2.95 vs. 15.97±7.33, numbers of crossing platforms (frequency/90 s): 0.70±0.95 vs. 2.70±0.48]; the entry angle was obviously greater than that of the normal control group [(47.94±4.68)°vs. (32.66±2.55)°, P < 0.05]. Compared with PAP mice model group, in Salvianolic acid group, the RTQ and number of crossing platform were significantly higher [RTQ (seconds): 13.57±1.86 vs. 8.27±2.95, number of crossing platforms (frequency/90 s):1.60±0.97 vs. 0.70±0.47], the entry angle was markedly smaller [(35.46±6.79)°vs. (47.94±4.68)°,P < 0.05]. The positive expression rate of APP and the protein expressions of CHOP, p-eIF2α in PAP mice model group were significantly higher than those in the normal control group [the positive rate of APP: (60.44±6.19)% vs. (21.05±5.87)%, CHOP protein expression (gray value): 3.09±0.07 vs. 1.46±0.09, p-eIF2αprotein expression (gray value): 0.98±0.09 vs. 0.47±0.06, all P < 0.01], the expression of PERK and p-PERK were lower than those in normal control group [PERK (gray value): 0.42±0.06 vs. 0.82±0.11, p-PERK protein expression (gray value): 0.98±0.09 vs. 0.64±0.10, both P < 0.01]; the positive expression rate of APP and protein expressions of CHOP, p-eIF2α in Salvianolic acid group were significantly lower than those in PAP mice model group [positive expression rate of APP: (33.09±10.33)% vs. (60.44±6.19)%, CHOP protein expression (gray value): 1.57±0.12 vs. 3.09±0.07, p-eIF2α protein expression (gray value): 0.80±0.07 vs. 0.98±0.09, all P < 0.01], while PERK and p-PERK expression were significantly higher than those in the model group [PERK (gray value): 0.89±0.12 vs. 0.42±0.06, p-PERK (gray value): 0.78±0.08 vs. 0.98±0.09, both P < 0.01]. Conclusion Salvianolic acid might work through the PERK-eIF2α-CHOP pathway to reduce the retention of APP in the hippocampus tissue of PAP dual-transgenic mice, thereby the learning ability of the mice is improved, and the progression of brain injury delayed.

Objective To explore the sample type and drug resistance characteristics of Streptococcus pneu-monia(Spn)isolated from pediatric patients in Guangzhou district,and their age distribution to offer instruc-tions for prevention and clinical treatment.Methods Spn isolates were cultured and identified according to the national standard procedure for clinical laboratory operation,followed by analysis of sample type and age dis-tribution of pediatric patients with positive isolates of Spn in Guangzhou Women and Children′s Medical Cen-ter from 2013 Jan 1st to 2015 Dec 31st,drug resistance status was determined by MIC test.Results Totally, 1 243 strains of Spn were isolated,which were mainly from pediatric patients under 1 year old(42.80%).Spn isolates were mainly isolated from respiratory tract(72.81%),ear secretions(15.37%),blood(5.63%),cere-brospinal fluid(3.06%)and hydrothorax(2.01%).For all Spn isolates,the resistance rate to erythromycin, tetracycline and sulfamethoxazole was especially high as 94.93%,85.76%,73.53% respectively,with relative high resistance to penicillin G(24.70%),amoxicillin(39.59%),ceftriaxone(24.05%),meropenem(22.85%) and cefotaxime(19.89%),low resistance to quinolone antibiotics(<10.00%),and no resistance to vancomycin and linezolid.Conclusion The major age group of children with Spn infection is infants under one year old in Guangzhou,clinicians should be serious about the high resistant rate of Spn to erythromycin,tetracycline and sulfamethoxazole,the significantly increased resistant rate to penicillin,amoxicillin and ceftriaxone.Clinicians should choose antibiotics rationally according to the characteristics of drug sensitivity for better treatment.