HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then transformed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to enhance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.
Cloning, Molecular ; Escherichia coli ; metabolism ; HIV Integrase ; metabolism ; HIV-1 ; physiology ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; Protein Binding ; Virus Replication

The general situation of the approved and concluded projects of National Natural Science Foundation of China in the field of processing Chinese Materia Medica in recent five years has been reviewed. The progresses and achievements of some projects have been summarized in accordance with research area such as the processing principle, the processing technology, quality evaluation, toxicity and safety evaluation, etc. The researchers and project support units of the funded projects have been analyzed, and the problems of the applications have been also summarized.
Biomedical Research ; economics ; organization & administration ; Chemistry, Pharmaceutical ; economics ; organization & administration ; China ; Financing, Organized ; economics ; organization & administration ; Humans ; Materia Medica ; economics ; Medicine, Chinese Traditional ; economics

Aged ; Female ; Humans ; Immunophenotyping ; Leukemia, Large Granular Lymphocytic ; classification ; immunology

0.05).Conclusion:Modified osteotomy can not improve operative effect.

Objective To establish an effective PCR assay for leptospirosis detection , and applicate the assay in tree shrew, mongolian gerbil and gray hamster .Methods Sequence of leptospira was obtained from the NCBI Genbank , and primers were designed based on the sequences .The positive amplified fragments were sequenced to verify the reliability of the method.The samples from tree shrew, mongolian gerbils and hamsters were tested using this PCR method .Results The PCR method for detection of leptospirosis was successfully established .The positive rate of Leptospira was 8.33% in 60 samples of conventional tree shrews , 100% in 104 samples of the conventional Mongolian gerbils , and 0% in 60 samples of clean gray hamsters.Conclusions The establishment of this PCR assay is useful in the detection of leptospirosis in tree shrew, mongolian gerbil and gray hamster .The results of our investigation of leptospira infection levels of the three new experimental animals may promote their application in biomedical research .

Objective To study the technological parameters of the purification process for active cons-tituents from Saussurea involucrata.Methods Using absorption and desorption rates of syringin,chologenic acid,and rutin as the primary screening indexes,four resins were selected,and the conditions of absorption and desorption were optimized for active constituents.Results Resin HPD400 gave good separation performance and was selected to purify the active constituents in S.involucrata.The optimum parameters were established as followings:4 BV(bed volume)sample extract whose pH value was adjusted to pH 2-3 by HCl was passed through the column with a flow rate of 1 BV/h,12 h later,the column was washed with 4 BV water,6 BV 30% ethanol and 4 BV 50% ethanol,respectively.The combined 30% and 50% ethanolic elutes were taken as object products.Conclusion The contents of active constituents were increased to about five times.Macroporous absorption resin HPD400 could be well used in separating and purifying the active constittuents from S.involucrata.

AIM: To investigate the incidence of trachoma in children aged 1 to 9y in Hainan Province and determine high-risk trachoma endemic and non-endemic areas in Hainan, and thus provide evidence for developing trachoma control and prevention therapy.METHODS:The areas of investigation were chosen on the basis of past literatures, expert interviews and survey on the spot.In 2013, Hainan Provincial Office of Blindness Prevention carried out the survey in 7 counties including Dongfang City, Wuzhishan City, Ledong County, Baisha County, Baoting County, Lingao County and Changjiang County.In these districts, 356 pupils including 192 boys and 164 girls were examined, their age ranging from 1 to 9 and their average age being 7 years old.The targeted students received the trachoma rapid assessment by the adoption of simplified trachoma classification system which was recommended by the World Health Organization.RESULTS: No case of active trachoma was found among the 356 students.CONCLUSION:The prevalence rate of trachoma in children under 9 years is less than 5% in Hainan Province.Active trachoma is not a public health issue in Hainan Province.

Objective To evaluate the applied value of multislice CT (MSCT) in the selection of living donor kidneys and excision methods.Methods Ninety living renal donors underwent MSCT assessment.The nonenhanced,arterial,venous and excretory phase examinations were performed.Using maximum intensity projection and volume rendering techniques for vascular imaging,two blinded radiologists independently analyzed and evaluated all MSCT images.According to the CT reconstructive images,radiologists and physicians selected the left renal or the right renal donors,and chose laparoscopic or open live donor nephrectomy.Results On the 90 cases of donors,78 donors underwent nephrectomy in the left kidney.Seventy-one left kidney donors having no significant variation received the routine laparoscopic live donor nephrectomy.Seven left kidney donors on both sides had relatively obvious anatomical variations such as accessory renal artery,multi-branch renal vein and renal vein in the back of the abdominal aorta,and they were subjected to the left kidney open donor nephre.ctomy.Other 12 donors having significant variation in the left kidney were given nephrectomy in the right kidney,and all of them received hand-assisted laparoscopic live donor nephrectomy.All intraoperative records of urine collection system and renal vascular anatomy were consistent with the preoperative evaluation of MSCT,and the accuracy was 100 ％.Two imaging experts in the evaluation of renal artery,renal vein and urine collection system showed good consistency.Nephrectomy was successfully performed on 90 cases of donors,and.postoperative recipients had no renal vein thrombosis and other vascular complications.Conclusion MSCT can provide accurate and valuable information for the selection of living donor kidneys and excision methods as a “one-stop” technique for the preoperative evaluation of living renal donors.

Objective To establish an effective PCR assay for detection of Bartonella, and application of this assay in tree shrew .Methods Sequence of Bartonella was obtained from NCBI Genbank .Three pairs of primers were designed based on this sequence .One pair of primers was determined through amplifying the major strains in China .Sixty tree shrew blood samples were tested with this PCR assay .The positive amplified fragments were sequenced to verify the reliability of this method .Results A PCR method for detection of Bartonella is successfully established , with a high specificity and the sensitivity was of 2.0 ×10 -5 μg/mL.Among the tested 60 blood samples , 15 positive cases were detected.Sequencing of the samples confirmed a 25%infection rate of Bartonella in the tree shrews, well consistent with the amplification results , and verified the applicability of this detection method .Conclusion The establishment of this method provides the basis for detection of Bartonella in tree shrew.

Objective: To investigate the effect of icariin on the proliferation, differentiation, and the mRNA expressions of Cbfαl, BMP2, BMP4 of rat osteoblasts. Methods: Primary rat osteoblastic cells were obtained by sequentia collagenase/trypsin enzyme digestion from calvarial bones of new born ( within 24 h) SD rats and were identified by Alkaline phosphatase and alizarin red staining. The passage 3-5cells were treated with icariin at the concentration of 0 mol/L, 10~(-8)mol/L, 10~(-7)mol/L, 10~(-6)mol/L,10~(-5)mol/L, 10~(-4)mol/L for 24 h, 48 h, 72 h, and the proliferation of the cells was measured by CCK-8assay. The proliferation index was detected by Flow Cytometry and the activity of alkaline phosphatase was determined by p-Nitrophenyl phosphate (pNPP) method after being treated with icariin at the concentration mentioned above for 48 h. The total cellular RNA was extracted 48 h after being treated with icariin at the concentration of 10~(-6)mol/L, and the expressions of Cbfα1, BMP2, BMP4 mRNA were examined by real-time PCR. Results: Icariin showed no effect on the proliferation of osteoblasts, but improved ALP activity. The Cbfα1, BMP2, BMP4 mRNA were significantly upregulated after icariin treatment. Conclusion: Icariin could promote the differentiation ability of rat osteoblasts through upregulating the Cbfα1, BMP2, BMP4 mRNA expressions.