Objective To construct the nucleic acid vaccine(DNA vaccine) plasmid of the gag gene of the prevalent HIV-1 strains in China so as to develop a therapeutic AIDS vaccine against the HIV-1 strains, and to investigate the immune responses induced by HIV-1 DNA vaccine in mice. Methods The recombinant expression vector pCI-neo GAG was constructed by inserting HIV gag gene into the eukaryotic expression vector pCI-neo, which was identified by restrictive enzymes (Xba Ⅰ/Sal Ⅰ) digestion analysis and DNA sequencing. Balb/c mice were immunized with pCI-neo GAG or pCI-neo. Their sera were collected for the assay of the titer of anti-HIV antibody and IFN-? level by ELISA, and their splenic cells were isolated for detecting antigen-specific lymphoproliferative responses and specific cytotoxic T lymphocyte(CTL) response by MTT assay and LDH assay, respectively. Results Restrictive enzymes digestion analysis and DNA sequencing results revealed that the recombinant expression vector pCI-neo GAG had been constructed successfully. Both the anti-HIV antibody titer and the IFN-? level of mice immunized with the pCI-neo GAG were higher than those of mice immunized with pCI-neo (P

Objective Clearing HIV provirus is the key to cure AIDS .The study was to construct the Tre enzyme eukaryotic expression vector and identify its function in specific recognition of loxLTR sequence in HIV provirus . Methods Tre gene was in-serted into eukaryotic expression vector pcDNA 3.1 gene recombination manipulation by genetic recombination techniques including gene synthesis , PCR, restriction enzyme digestion and ligation .EGFPpA-LoxLTR sequence was inserted into pmCherry-N1 vector and was tested by restriction enzyme digestion , PCR and sequencing .Constructed vectors were electroporated into HeLa cells , then using fluorescence microscopy to observe fluorescence intensity changes . Results PCR, restriction enzyme digestion , electrophoresis and sequencing confirmed that Tre enzyme eukaryotic expression vector had been constructed successfully , and it could specifically recog-nize and cut loxLTR sequence after being transfected into Hela cells . Conclusion Constructed Tre enzyme eukaryotic expression vector can be expressed in Hela cells and specifically recognize loxLTR sequence , which has prepared the experimental ground for fur-ther studies of clearing HIV provirus .

Objective:To construct the eukaryotic expression vector of interleukin-18 gene and carry out analysis of pVAX1-IL-18 sequence . Methods:The recombinant eukaryotic expression vector pVAX1-IL-18 was constructed by inserting interleukin-18 gene into the eukaryotic expression vector pVAX1 with molecular cloning technique . It was confirmed by restrictive enzymes(BamHⅠ/ EcoRⅠ) digestion and analysis of DNA sequencing . Similar nucleotide sequences encoding IL-18 of pVAX1-IL-18 were looked up by Blast means in NCBI GenBank. Results:Restrictive enzymes digestion analysis and DNA sequencing results revealed that the interleukin-18 gene was cloned into the eukaryotic expression vector pVAX1 successfully .The sequence of encoding IL-18 of pVAX1-IL-18 was exactly the same as the reported sequence of encoding human IL-18 in GenBank . Conclusion: The recombinant eukaryotic expression vector pVAX1-IL-18 has been constructed successfully, which lay the foundation for pVAX1-IL-18 as a genetic adjuvant of DNA vaccine.

To evaluate the inhibitive effect of plant protein MAP30 (momordica anti-HIV protein of 30kDa), AZT (3′-azido-2′,3′-dideoxythymidine) , ACV (acyclovir) and IFN-?2a (interferon-?2a) against HIV-1 in vitro, MT4 was used as the target cell and the inhibitive effects of these drugs on HIV-1 P24 expression were investigated by ELISA. The inhibitory effect of these drugs on HIV-1-induced cytopathy was also evaluated. The 50% inhibition concentration (IC 50) of MAP30 was 0.9?mol/L. In comparison, the IC 50 for AZT , a commonly used anti-HIV drug, was 0.7?mol/L. The cytopathic effect induced by HIV-1 was also inhibited by MAP30 and AZT. ACV and IFN-?2a showed little effect on HIV-1. All these results strongly indicated that plant protein MAP30 could obviously inhibit HIV-1 replication in vitro.

Objective To explore the possibility of human neutrophil peptide 1 (HNP1) gene engineering, we construct the eukaryotic expression vector carrying HNP1 gene. Methods With RNA extracted from human neutrophil cell as template, cDNA encoding mature HNP1 was amplified by RT-PCR, and then it was inserted into vector pMD18-T. After restriction endonuclease digestion and DNA sequencing confirmation the gene was subcloned into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO to construct a recombinant expression plasmid pcDNA3.1/V5-His-TOPO/HNP1, then the recombinant plasmid was transfected into COS-7 cells by lipofectamine, and the expressed product was identified by biotin-avidin enzyme-linked immunosorbent assay ( BA-ELISA). Results The sequence of HNP1 completely matched those published in GenBank, thus eukaryotic expression vector pcDNA3.1/V5-His-TOPO/HNP1 was constructed correctly. The ELISA results showed that the eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO/HNP1 could temporarily express HNP1 in COS-7 cells. Conclusion The successful construction and expression of pcDNA3.1/V5-His-TOPO/HNP1 pave the way for the stable expression HNP1 in mammalian engineering cells.

Objective: To assay the content and stability of vitamin C(VC) in serum by a simple, specific method. Methods: Serum samples were extracted by metaphosphoric acid, and analyzed on a column C 18 of HPLC with DAD detector at 234 nm. Mobile phase was 100 mmol/L KH 2PO 4, pH3.00. The flow rate was 1 ml/min. Results: Under this condition, the retention time of VC was 5.76 min. The within run and between run reproducibilities tests were 0.42% and 2.11% respectively, and the recovery rate ranged from 82% to 88.5%. Stored at -70 ℃ and compared to that of its metaphosphoric acid extract, serum VC content was stable only for two days, and then decreased rapidly, whereas it did not decrease in metaphosphoric acid extract until the fifth day. Conclusion: A simple, sensitive and reliable method of HPLC with DAD detector is established for measuring VC in either serum or fruit, such as orange juice. VC should be determined immediately or should be extracted with metaphosphoric acid as soon as possible, then the extracts are kept at -70 ℃, and analysed within 5 d.

BACKGROUND:Currently, surgical treatment for Sanders III type, IV type fractures to restore damaged subtalar surface of the calcaneus and calcaneal shape, in order to reduce the incidence of traumatic arthritis has reached a consensus, and whether bone grafting is selected during surgery for calcaneal fractures has been a controversial issue. OBJECTIVE:To observe the clinical effect of Genex synthetic bone transplants and locking plate on comminuted calcaneal fractures. METHODS:Twenty-one cases of Sanders III type, IV type calcaneal fractures were retrospectively analyzed, including 16 males and 5 females, aged 22 to 55 years. After fracture reduction, a dough-like Genex bone graft was implanted into the defect region via lateral“L”shaped approach, and then the lateral wal of the bone was reset fol owed by internal fixation with the pre-curved locking plate. Fol ow-up observation was performed for fracture healing, Bolher angle and the Maryland Foot Score. RESULTS AND CONCLUSION:Total y 21 patients were fol owed up for 8-16 months. The fractures were healed without displacement, col apse and rejection. The bone graft was degraded within 6 months and completed absorbed after 1 year. According to Maryland Foot Score, the excellent rate was up to 86%, and the Bolher angle was increased from an average preoperative (5.3±3.35)° to postoperative (24.3±1.06)°. Genex artificial bone meal is a biomaterial that can be completely absorbed, has good plasticity and strong supporting force, and it is able to ful y fil bone defects, be easy to fracture reduction, induce bone formation, and promote fracture healing.

Objective To investigate the metastasis mechanism by observing morphological distributions of lymphic vessels in peripheral areas of the different development of uterine cervix cancer. Methods Cancer tissues from the center, peripheral and normal areas of uterine cervix cancer respectively were collected. The paraffin sections and semithin sections which were stained with HE were applied to those tissues for exploring the configurations and distributions of lymphic vessels of the cancer under a microscope. And the ultrathin sections were applied to those tissues for exploring under a electronic microscope. Results Under the microscope, the basement membrane had been destroyed by cancer cell, which continued to infiltrate interstitial tissue. Lymphic vessels were increased and dilated in peripheral areas of uterine cervix cancer than those in normal areas. Moreover, the walls of lymphic vessels were hazy and broken. Conclusion The increase and morphologic changes of lymphic vessels in peripheral areas of uterine cervix cancer will play an important role in lymphatic metastasis.

Objective To research and evaluate measuring Toric intraocular lens (Toric IOL) alignment by iTrace aberration without mydriasis.Methods Forty-five eyes of 35 patients underwent phacoemulsification in Tianjin Medical University Eye Hospital from June 2015 to February 2016 were enrolled.Follow-up and iTrace aberration examination were performed at postoperative 1 week.The internal optics aberration astigmatism axis was transformed into postoperative Toric IOL alignment.The result and the Toric IOL alignment measured by tradition slitlamp method were compared by linear correlation and difference.Results At postoperative 1 week,the uncorrected distant visual acuity and corrected distant visual acuity were (0.19 ± 0.12)LogMAR and (0.10 ±0.09) LogMAR.The UCVA was 20/40 or better in 42 eyes (93.3％).The mean IOL misalignment measured by slitlamp was (3.13 ± 2.86) degrees (ranged 0-9 degrees) and by the iTrace aberration was (4.44 ± 3.42) degrees(ranged 0-13 degrees),there was statistical significant difference (t =-2.321,P =0.025).The mean difference in the error of the Toric intraocular lens alignment measured by iTrace aberration and the slitlamp was (3.67 ± 3.59) degrees (ranged 0-14 degrees).The results showed that there was less than 5 degrees of difference between the two methods in 32 eyes (71.1％),locate 5 to 10 degrees in 9 eyes (20％),more than 10 degrees in 4 eyes (8.9％).The correlation between the 2 methods showed significant linear relationship (r =0.926,P ＜ 0.01).Conclusion Using iTrace aberration can accurately measure Toric intraocular lens alignment without mydriasis,the result has some reference value.