Preparation of rabbit monoclonal antibody against cGMP and development of competitive ELISA for cGMP / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare rabbit monoclonal antibody (RabMab) against guanosine 3', 5'-cyclic monophosphate (cGMP) and to develop a competitive ELISA for the detection of cGMP.</p><p><b>METHODS</b>New Zealand white rabbits were immunized with synthesized cGMP-keyhole limpet hemoeyanin (cGMP-KLH) to prepared a RabMAb with monoclonal antibody technique of Epitomics. A competitive ELISA kit was produced with cGMP RabMAb. The specificity, the precision and the recoveries of the method were determined.</p><p><b>RESULTS</b>The RabMAb with high sensitivity towards cGMP were prepared with an antibody timer of 3.1 ng/mL and 50% inhibitive concentration (IC50) of 12.57 ng/mL. The cGMP RabMAb had 33% cross-reactivity to inosine 3', 5'-cyclic monophosphate (cIMP) and little or no cross-reactivity to other compounds. A competitive ELISA was developed for detection of cGMP. The range of detection was 0~120 ng/mL with a minimal limit of 1.95 ng/mL. The recovery of assay was 89%~103%. The inter-assay and intra-assay coefficient variations were below 11.68% and 13.85%, respectively.</p><p><b>CONCLUSION</b>The RabMab against cGMP with high affinity and high specificity has been generated successfully, and a competitive ELISA for detection of cGMP has been developed with the prepared cGMP RabMAb.</p>

Combined use of compound nanxing pain paste with blood-promoting and diuretic chinese herbal medicines in treatment of chronic knee synovitis / 中国骨伤

<p><b>OBJECTIVE</b>To observe the clinical efficacy of combined use of Compound Nanxing Pain Paste with blood-promoting and diuretic Chinese herbal medicines in treatment of chronic knee synovitis.</p><p><b>METHODS</b>From October 2008 to March 2011, 120 patients with chronic knee synovitis were equally divided into three groups: oral treatment group, external treatment group and combined treatment group. Patients in oral treatment group had a mean age of (56.58 +/- 5.47) years and a course of disease about (7.35 +/- 2.59) months,involving the left knee in 18 cases,the right knee in 17 cases and both knees in 5 cases, who were administered orally with blood-promoting and diuretic Chinese herbal medicines composed of Wu Ling San and Tao Hong Si Wu Decoction. Patients in external treatment group had a mean age of (56.25 +/- 6.35) years and a course of disease about (7.68 +/- 2.76) months,involving the left knee in 16 cases, the right knee in 20 and both knees in 4 cases, who were treated externally with Compound Nanxing Pain Paste. Patients in combined treatment group had a mean age of (55.65 +/- 4.49) years and a course of disease about (7.50 +/- 3.36) months, involving the left knee in 16 cases, the right knee in 18 and both knees in 6 cases, who were given both oral and external treatments. The clinical effects and knee function of the three groups were assessed and compared.</p><p><b>RESULTS</b>Finally, a total of 118 patients were included in the result analysis, including 40 patients in oral treatment group, 39 patients in external treatment group and 39 patients in combined treatment group. In combined treatment group, 27 patients were clinically cured, 9 were improved, and 3 patients were ineffective vs. 15, 16 and 8 patients in external treatment group, and 13, 16 and 11 in oral treatment group. The overall effective rate of combined treatment group was better than that of oral group and external treatment groups (P < 0.05). The function comprehensive score of knee joints in combined treatment group was better than that of oral treatment group,while the function comprehensive score of knee joints in oral treatment group was better than that in external treatment group in terms of demand for assistance, stair-climbing ability, lameness, and swelling pain.</p><p><b>CONCLUSION</b>Combined use of oral blood-promoting and diuretic Chinese herbal medicines with Nanxing offers a good therapeutic effect on chronic knee synovitis in relieving pain, reducing swelling and improving joint function,while Nanxing could also reduce pain and improve joint function to a certain extent.</p>

Codon optimization of recombinant staphylococcal enterotoxin O enhances the expression level in Escherichia coli / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To enhance the expression level of staphylococcal enterotoxin O (SEO) by optimization of rare codons.</p><p><b>METHODS</b>The gene of mature SEO (His-tag included) was cloned to pET28a, and 15 rare codons on the gene were optimized by PCR technology. These recombinant plasmids then were transformed into E.coli BL21(DE3), respectively. After IPTG induced, the expression levels of those mutants were analyzed by SDS-PAGE. The proteins were purified and their bioactivities were determined.</p><p><b>RESULT</b>After the optimization of rare codons, the expression levels were increased from 7.49% to 19.8% in total cell proteins. The optimized SEO had bioactivity to stimulate the proliferation of murine lymphocytes, which was equivalent to that of non-optimized SEO in vitro.</p><p><b>CONCLUSION</b>Optimization of rare codons can enhance the expression of SEO effectively.</p>

CYP2D6*1, CYP2D6*10 co-expressed with CYPOR in Bac-to-Bac expression system and activity determination / 药学学报

CYP2D6 is an important drug-metabolizing enzyme. The polymorphism of CYP2D6 leads to metabolism difference and the different reactions of drugs in the individuals and different races are normal phenomenon in clinical medication. CYP2D6*10 is an important subtype in Asian people and 51.3% Chinese are classified with this subtype. To obtain recombinant active CYP2D6*1/CYP2D6*10 in baculovirus system by optimizing coexpression with CYPOR, and detect their activity to catalyze dextromethorphan, three recombinants pFastBac-CYP2D6*1, pFastBac-CYP2D6*10 and pFastBac-CYPOR were constructed and transformed into DH10Bac cell to obtain the recombinant Bacmid-CYPOR, Bacmid-CYP2D6*1 and Bacmid-CYP2D6*10. And then the recombinant CYP2D6*1 and CYP2D6*10 virus were obtained by transfecting Sf9. Then homogenate protein activity was determined with dextromethorphan as substrate. The multiple of infection (MOI) and its ratio of recombinant CYP2D6 virus to CYPOR virus were adjusted by detecting the activity of the homogenate protein. The Km and Vmax are 26.67 +/- 2.71 micromol x L(-1) (n=3) and 666.7 +/- 56.78 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*1 to catalyze dextromethaphan. The Km and Vmax are 111.36 +/- 10.89 micromol x L(-1) (n=3) and 222.2 +/- 20.12 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*10 to catalyze dextromethorphan. There is significant difference between CYP2D6*1 and CYP2D6*10 for Vmax and Km (P < 0.01). The clearance ratio of CYP2D6*1 is 25.0 and the clearance ratio of CYP2D6*10 is 2.0. The expressed CYP2D6*1 and CYP2D6*10 are useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro, which are benefit to understand individual metabolism difference.

The role of ADME evaluation in translation research of innovative drug / 药学学报

New Chemical Entities (NCEs) development is a systematic long-term project that involves multiple disciplines. The translation research will help to build an advanced R&D system from the basic laboratory research, preclinical studies and clinical evaluation to clinical application of drug, for the purpose of shortening the R&D cycle and accelerate the launch of new drugs. In new drug R&D and its clinical application, drug disposition (absorption, distribution, metabolism, excretion, ADME) properties are important criteria for assessing drug-likeness of candidates. ADME evaluation of NCEs plays an important role in the translation research throughout innovative drug R&D process. Therefore, ADME evaluation at the early stage of drug design and development will be helpful to improve the success rate and reduce costs, and further access to safe, effective drugs.

Determination of cotinine, phenylglyoxylic acid and mandelic acid in human urine by GC/MS / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a GC/MS method for analysis of cotinine (COT), phenylglyoxylic acid (PA) and mandelic acid (MA) in human urine.</p><p><b>METHODS</b>Human urine samples were extracted by CCl(3) and derivatized with MSTFA after dried completely. The contents of COT, PA and MA were measured by GC/MS method with DB-5MS capillary column and EI ion-source.</p><p><b>RESULT</b>The calibration curves for COT in urine samples were linear over the concentration ranges of 0.0002 approximately 3.5 microg ml(-1), while PA and MA were both of 1.25 approximately 160 microg ml(-1). The limits of quantification were 0.0002 microg ml(-1), 1.25 microg ml(-1) and 1.25 microg ml(-1) for COT, PA and MA, respectively. The assay recoveries for COT, PA and MA ranged from 89.53% approximately 102.4%, 84.88% approximately 91.46% and 83.46% approximately 13.6%, respectively.</p><p><b>CONCLUSION</b>The established method can detect cotinine, phenylglyoxylic acid and mandelic acid simultaneously, which would be used in routine assessment and monitoring of the internal exposure to nicotine and styrene in human body.</p>

Determination of beta-estradiol, bisphenol A, diethylstilbestrol and salbutamol in human urine by GC/MS / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a GC/MS method for analysis of beta-estradiol (beta-E2), bisphenol A (BPA), diethylstilbestrol (DES) and salbutamol (SAL) in human urine.</p><p><b>METHODS</b>Human urine samples were extracted by cleanert PCX and cleanert PEP cartridges; and derivatized after dried completely. beta-E2, BPA, DES and SAL in the extracts were measured by GC/MS method with DB-5MS capillary column and EI ion-source.</p><p><b>RESULT</b>The calibration curves for beta-E2 in samples were linear over the concentration ranges of 1 approximately 300 ng ml(-1), for BPA were 1 approximately 200 ng ml(-1), for DES were 2 approximately 300 ng ml(-1) and for SAL were 0.01 approximately 1.2 microg ml(-1). The limits of detection were 0.15 ng ml(-1), 0.19 ng ml(-1), 0.23 ng ml(-1) and 1.0 ng ml(-1)for beta-E2, BPA, DES and SAL, respectively. The assay recoveries for beta-E2, BPA, DES and SAL ranged from 93.4 % approximately 110.5 %, 84.7 % approximately 104.9 %, 87.0 % approximately 105.4 % and 81.8 % approximately 96.8 %, respectively.</p><p><b>CONCLUSION</b>The established GC/MS method can detect beta-E2, BPA, DES and SAL in urine samples simultaneously, which can be used in routine assessment and monitoring of beta-E2, BPA, DES and SAL in human body.</p>

Purification of fusion protein HSA/IL1ra and its bioactivity / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To purify the recombinant human serum albumin fusion protein with interleukin 1 (HAS/IL1ra) and to detect the bioactivity of the fusion protein.</p><p><b>METHODS</b>The recombinant HAS/IL1ra protein was purified by affinity chromatography and ion exchange chromatography, the bioactivity of the fusion protein was detected by IL1-induced A375 S2 cell killing.</p><p><b>RESULT</b>The purity of the fusion protein was at least 98 % as assessed by HPLC and the protective effect from the IL1-induced A375 S2 cell killing was similar to natural IL1ra.</p><p><b>CONCLUSION</b>The purified recombinant HAS/IL1ra protein in this study has a satisfactory bioactivity.</p>

Preparation and identification of monoclonal antibodies to staphylococcal enterotoxin I / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare and identify monoclonal antibodies against staphylococcal enterotoxin I (SEI).</p><p><b>METHODS</b>Spleen cells obtained from mice immunized with the SEI protein were fused with the myeloma cells (SP2/0). Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) and the stable monoclonal hybridomas were isolated by limiting dilution at least three times. The characters of purified monoclonal antibodies were identified by indirect ELISA and Western blotting.</p><p><b>RESULT</b>The monoclonal antibodies secreted by two hybridomas 8F7 and D8 belonged to IgG(2b) and IgG(1) subtypes. Both had high titer and specificity with no cross reaction to SEG, SEE and SEC.</p><p><b>CONCLUSION</b>The monoclonal antibodies against SEI has been successfully prepared and identified in this study.</p>

Peptide bond scission of staphylococcal enterotoxin C2 and related factors / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the limited digestion of recombinant staphylococcal enterotoxin C2 (SEC2-His)in different conditions.</p><p><b>METHODS</b>The purified recombinant SEC2-His was treated with different reagents and the cleavage of rSEC2 molecule was observed by SDS-PAGE.</p><p><b>RESULT</b>The cleavage occurred in positions Cys93-Cys110 of the disulfide loop. Complete auto-cleavage of recombinant SEC2 was observed in solution at 37degrees within 24 hrs, and that was accelerated under alkaline conditions. The auto-cleavage of the recombinant protein was inhibited in the presence of beta-ME (2%), PMSF (5-10 mmol/L), imidazole (1 mol/L) or crude E.coli lysate. Non-specific degradation of recombinant SEC2 was promoted with the increasing of the concentration of H(2)O(2).</p><p><b>CONCLUSION</b>The recombinant SEC2-His is broken down in special site of protein, which may be associated with the protein structure.</p>

Alternatively spliced variants of constitutive androstane receptor in liver of mouse / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the existence of alternatively spliced variants of constitutive androstane receptor (CAR) in liver of mouse.</p><p><b>METHODS</b>The nucleotide from liver of mouse was purified and the CAR cDNA was amplified by PCR. The fragments of CAR cDNA were cloned to T vector and sequence analysis was performed.</p><p><b>RESULT</b>Various spliced variants of CAR in liver mouse were confirmed by DNA sequencing.</p><p><b>CONCLUSION</b>There are alternatively spliced variants in CAR, which are located in the ligand binding sequence of CAR.</p>

Construction, expression and characterization of recombinant fusion protein HSA-PTH (1-34) in Pichia pastoris / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To obtain recombinant fusion protein HSA (human serum albumin)-PTH(1-34) in Pichia pastoris.</p><p><b>METHODS</b>HSA and PTH(1-34) cDNA were obtained with PCR and the DNA segments were cloned into vector pPIC9 with linker. The linearized plasmids were transformed GS115 competent cells treated with LiCl, and mut+ transformants were screened on MD plate. With AOX promoter and alpha-MF signal sequences leading, fusion protein was expressed in GS115. PCR and SDS-PAGE were employed to confirm the integration and expression of HSA-PTH(1-34). The fusion protein was identified by Western blotting and classical adenylate cyclase assay.</p><p><b>RESULT</b>The PCR results showed that the gene of HSA-PTH(1-34) was integrated into GS115 genome. Western bolt approved the existence of two domains of HSA and PTH(1-34). The bioactivity assay in rabbit cortical membranes indicated that HSA-PTH (1-34) activated adenylate cyclase, but the activity was lower than that of the synthetic PTH(1-34).</p><p><b>CONCLUSION</b>Active fusion protein HSA-PTH (1-34) is successfully expressed in Pichia pastoris.</p>

Inhomogeneous expression of fusion protein HSA/IL1ra in Pichia pastoris / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the causes and influencing factors of heterogeneity of HSA/IL1ra fusion protein expression in Pichia pastoris.</p><p><b>METHODS</b>The heterogeneity of HSA/IL1ra fusion protein expressed in Pichia pastoris was studied by removing glycosylation and inhibiting glycosylation, as well as by different ways of fusion, different clones, and different expression host.</p><p><b>RESULT</b>Glycosylation caused expression heterogeneity of fusion protein, but in SMD1168 and some GS115 clones there was no this phenomenon.</p><p><b>CONCLUSION</b>The expression heterogeneity of HSA/IL1ra fusion protein in Pichia pastoris is due to the glycosylation, and different ways of fusion, different clones, different expression host also have some impact.</p>

Coexpression of PXRLBD with SRC88 and construction of equilibrium dialysis model of screening PXR ligands / 药学学报

The aim of this study was to obtain the soluble protein of human pregnane X receptor ligand binding domain (PXRLBD) through the coexpression of PXRLBD and 88 amino acids of steroid receptor coactivator-1 (SRC88) and apply the protein to constructing a new model of screening PXR ligands. Expression plasmid of pETDuet-1-SRC88-PXRLBD was constructed and transformed into Escherichia coli Rosetta (DE3) to coexpress PXRLBD and SRC88 via induction by IPTG at low temperature. Then an equilibrium dialysis model was constructed to study the interaction between PXRLBD and drugs including clotrimazole and dexamethasone, using HPLC as the analysis method. The results showed that the soluble protein of PXRLBD was obtained and the HPLC data indicated that clotrimazole bound to PXRLBD, while dexamethasone did not bind to PXRLBD, which indicated the successful establishment of a new method for studying the interaction between PXR and drugs. The new method may be useful in the screening of PXR ligands in vitro.

Preparation and application of antibody against staphylococcal enterotoxin C2 / 药学学报

The filtrate of Staphylococcus aureus culture has been used in an ampule form named as staphylococcal enterotoxin C injection for cancer therapy in clinic for ten years in China and proved to be effective. The active constituent of three kinds of injections is claimed to be staphylococcal enterotoxin C2 (SEC2), and the content of SEC2 is used as quality control. However, the correct content of SEC2 was not known and the relative amount of SEC2 was very low because of the complicated components of the filtrate. In this research, we established a proper ELISA system for the detection of SEC2 in staphylococcal enterotoxin C injection, which will improve the quality control of the injection. We produced and identified polyclonal and monoclonal antibodies of SEC2 and established BA-ELISA method based on the method of sandwich ELISA. It was found that the BA-ELISA method had good specificity, sensitivity and reproducibility, and being able to detect SEC2 at concentration from 2 to 20 ng x mL(-1), with an average CV value of 5.08%. The SEC2 content in staphylococcal enterotoxin C injection was calculated. There is some difference between the actual and labeled contents in the injections.

Expression and bioactivity of the cloned staphylococcal enterotoxin O / 药学学报

This study is to clone the gene of staphylococcal enterotoxins O, obtain recombinant protein (rSEO) and investigate its activity on mice lymphocyte. Staphylococcus aureus O gene is cloned into GST gene fusion vector pGEX-4T-1. The resultant plasmid pGEX-4T-SEO was used to transform E. coLi BL21, where the GST-SEO fusion protein was expressed efficiently. Then SEO was purified by Glutathione Sepharose 4B affinity column and digested with thrombin. The bioactivity of SEO was analyzed by MTT assay on mice lymphocyte and tumor cells. The nucleotide sequence was confirmed to code for the protein correctly, and soluble SEO was expressed efficiently in E. coli BL21 with pGEX-4T-SEO. The protein purified by affinity chromatography resulted to be one single band by SDS-PAGE detection. The MTT assay of the purified rSEO demonstrated that its abilities of stimulating T cells and inhibiting the proliferation of K562, K562-ADM and B16 cells were equivalent to that of SEC in vitro. The expression plasmid pGEX-4T-SEO was constructed and the recombinant superantigen was expressed successfully, which may provide a foundation for the further research of the anticancer activity of SEO.

Pharmacokinetics of PEG-rhG-CSF and rHSA-hG-CSF in mice determined with ELISA / 药学学报

Double antibody sandwich-type ELISA was used to detect rhG-CSF in serum to study the pharmacokinetics of rhG-CSF, PEG-rhG-CSF and rHSA-hG-CSF in mice and to confirm that PEGlyation and albumin fusion of rhG-CSF technology can prolong half-life of G-CSF. Pharmacokinetic parameters were calculated with 3P87 software. T1/2 s of rhG-CSF, PEG-rhG-CSF and rHSA-hG-CSF are 2. 1 , 14.2 and 10. 6 h, respectively. T1/2 s of PEG- rhG-CSF and rHSA-hG-CSF are 7, 5 times than T1/2 s of rhG-CSF, respectively. Tpeak s of PEG-rhG-CSF and rHSA-hG-CSF are 15, 13 times than Tpeak of rhG-CSF, respectively. The result of ELISA indicates that PEGlyation and albumin fusion of rhG-CSF technology can prolong half-life of G-CSF.

Stereoselective glucuronidation of carvedilol by Chinese liver microsomes / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To study the stereoselective glucuronidation of carvedilol (CARV) by three Chinese liver microsomes.</p><p><b>METHODS</b>The metabolites of CARV were identified by a hydrolysis reaction with beta-glucuronidase and HPLC-MS/MS. The enzyme kinetics for CARV enantiomers glucuronidation was determined by a reversed phase-high pressure liquid chromatography (RP-HPLC) assay using (S)-propafenone as internal standard after precolumn derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosylisothiocyanate.</p><p><b>RESULTS</b>Two CARV glucuronides were found in three Chinese liver microsomes incubated with CARV. The non-linear regression analysis showed that the values of K(m) and V(max) for (S)-CARV and (R)-CARV enantiomers were (118+/-44) micromol/L, (2 500+/-833) pmol/(min.mg protein) and (24+/-7) micromol/L, (953+/-399) pmol/(min.mg protein), respectively.</p><p><b>CONCLUSION</b>These results suggested that there was a significant (P<0.05) stereoselective glucuronidation of CARV enantiomers in three Chinese liver microsomes, which might partly explain the enantioselective pharmacokinetics of CARV.</p>

Expression and bioactivity analysis of staphylococcal enterotoxin C2 / 药学学报

<p><b>AIM</b>To clone the gene of staphylococcal enterotoxin C2 and express it in the form of a soluble fusion protein in E. coli. Then the activation of SEC2 on mice lymphocyte and its lethal effects on tumor cells were studied.</p><p><b>METHODS</b>Staphylococcus aureus SEC2 gene was cloned into GST gene fusion vector pGEX-4T-1. The resultant plasmid pGEX-4T-SEC2 was used to transform E. coli BL21, where the GST-SEC2 fusion protein was expressed efficiently. The rSEC2 protein was purified with Glutathione Sepharose 4B affinity column and digested with thrombin. The in vitro culture system was utilized to observe the activation of the SEC2 on mice lymphocyte and the lethal effects on tumor cells of the activated mice lymphocyte.</p><p><b>RESULTS</b>The proper gene of SEC2 was cloned and purified rSEC2 was obtained. The MTT results indicated that rSEC2 have strong ability to stimulate mice lymphocyte to proliferate with a dose-dependent manner. With the proliferation of mice splenic lymphocyte, rSEC2 has a strong lethal effect on tumor cells B16, K562 and K562-AD.</p><p><b>CONCLUSION</b>In this study, the gene of SEC2 was cloned and the rSEC2 protein was obtained, which had strong lethal effect on tumor cells B16, K562 and K562-AD.</p>

Mapping of regulatory domain of T-protein from Escherichia coli / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To map the regulatory domain of Escherichia coli T-protein.</p><p><b>METHODS</b>Fragmentation cloning was employed in cloning of 11 fragments from T-protein. The regulatory activity of each fragment was determined respectively.</p><p><b>RESULTS</b>The regulatory domain of T-protein was located in the C-terminal 270 amino acids, which was the same location as PDH domain.</p><p><b>CONCLUSION</b>T-protein has no independent regulatory domain.</p>