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Objective:To single amino acid mutation of the full human scFvs against TSLP to enhance its affinity.Methods: The specific scFvs against TSLP was screened in our previous study and here the three-dimensional structures of TSLP and anti-TSLP scFvs were simulated by Discovery Studio system,then the molecular docking was made.The amino acids of binding epitope were randomly mutated and the mutated amino acids were selected which could remarkably improve the affinity of scFvs.The primers were designed based on the sequence of mutation amino acids and the scFv sequences were mutated by the overlapping extension PCR.The DNA of mutated scFvs was ligated with the expression vector pLZ16 and transformed into E.coli DH5αF′.Then the scFvs were expressed and the scFvs with improved affinity were selected by ELISA and BIAcore.Results: The five scFvs with single amino acid mutation were screened out by DS system,which could elevate the affinity of scFvs.The mutated anti-TSLP-scFvs were amplified by PCR,which size was about 1 000 bp.The mutated scFvs with correct sequence were expressed,and the mutated scFvs with improved affinity were detected by ELISA and BIAcore.The affinity of selected mutated scFv (M4) has been about 10 times higher than the scFv nonmutation.Conclusion: The affinity of anti-TSLP-scFv has been improved successfully.
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Objective:To select specific and neutralizing scFv against IL-13 and soluble expression and identification them.Methods:In our previous study the scFv library were constructed,and here the scFv library was enriched and the positive scFv were screened from the enriched scFv library for three round.The specific positive scFvs with better affinity were ligated with expression vector for expression and identification.Results:After three rounds of enrichment,30%of clones were positive.The two specific scFvs with better affinity and neutralization were selected from almost 500 clones and then ligated with expression vector LZ16 for soluble ex-pression.The expressed scFvs were identified by western blot and biomolecular interaction analysis.Conclusion: The specific scFvs against IL-13 with better affinity and neutralization had been selected successfully.
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Objective:Expression and purification of the α-HL of Staphylococcus aureus as antigen for making full human anti-α-HL antibody later ,providing of new treatment for Staphylococcus aureus infection.Methods:The total RNA of Staphylococcus aureus was extracted and the cDNA of α-HL was amplified by RT-PCR.The DNA of α-HL and pCold-TF plasmid was digested and ligated by T4 ligase and then transformed into E.coli TOPO 10.The recombinant plasmid α-HL/pCold-TF which verified by sequencing was trans-formed into E.coli BL21 for expression.The expression products was identified by SDS-PAGE and Western blot.Results: The size of amplified cDNA of α-HL was about 900 bp and the expressed soluble fusion protein of α-HL was about 90 kD(including the molecular chaperone in the vector ) after inducing expression for 24 h at 15℃.The Western blot results showed that the expressed protein was the fusion protein of α-HL.The purified α-HL was injected into BABL/c mice for making antiserum.The results showed that the antiserum had good binding activity with Staphylococcus aureus and the titer was greater than 10 000 times.Conclusion: The α-HL of Staphylococcus aureus was successfully cloned and the soluble fusion protein of α-HL was successfully expressed.
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Objective Construction of human IL‐4 recombinant expression vector and then conduct the expression ,purification and identification of human recombinant IL‐4 .Methods the open reading frame of IL‐4 was amplified by nest PCR with total RNA from PBMC of healthy volunteer .And then the amplified IL‐4 was inserted into pET101/D‐TOPO ,transformed into BL21 ,ex‐pressed ,purified and indentified .Results The size of amplified open reading frame of IL‐4 was about 460 bp and the sequence was correct .After transformed into BL21 ,the IL‐4 clone with higher expression level was selected by selection of different clones insert‐ed with IL‐4 and the size of expressed ,purified IL‐4 was about 28 × 103 .Western blot results showed that the size of single band was identical with the expected protein .Conclusion Human IL‐4 recombinant protein was got successfully .
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The expressions of vascular endothelial growth factor(VEGF)and pigment epithelium derived factor(PEDF)in the tubulointerstitium were determined by immunohistochemistry in the streptozotoein-induced diahetic rats.The mRNA expressions of VEGF and PEDF in normal rat kidney (NRK) were measured by using RT-PCR under different high glucose conditions.The results showed that VEGF expression increased and PEDF expression decreased in the kidney tissue of the streptozotocin-induced diabetic rats.Hiish glucose increased VEGF expression and decreased PEDF expression in N RK cells in a time-and dose-dependent manner.The unbalanced expressions of VEGF and PEDF may play an important role in the occurrence and progression of diabetic nephropathy.
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Objective To investigate the relation between the expression of vascular endothelial growth factor (VEGF) and type Ⅳ collagen and the renal lesion of diabetes mellitus in diabetic rats. Methods Wistar rats were divided at random into normal control group(NC group) and diabetic model group(DM group), each group containing 20 animals. The rats models of diabetic mellitus were induced by streptozotocin(STZ). At the 8th week after induction, the expression levels of VEGF and type IV collagen in the kidney tissues in the two groups of rats were detected using immunohistochemistry. At the same time, the renal function of the rats was measured. Results At the 8th week,the expression levels of VEGF and type IV collagen of kidney tissues in DM group was much higher than those in NC group(P
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Objective To study the effect of valsartan on expression of MCP-1 in the myocardium of diabetic rats,to investigate the protective effect of valsartan on the myocardium of diabetic rats.Methods 30 rats were divided into at random:normal control group(NC group),diabetic model group(DM group),diabetic model plus valsartan therapy group(DV group).Diabetic rats were induced by STZ,at 8th week,expression of MCP-1 in the myocardium of diabetic rats,and diabetic rats treated with valsartan was detected respectively by using immunohistochemistry.Results At 8th week,the expression of MCP-1 of DM group was much higher than in both DV group and NC group(P
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OBJECTIVE:To study the pharmacokinetics of compound amlodipine besylate/atorvastatin calcium tablets(CABACT) following oral administration of single dose vs. multiple doses in healthy volunteers. METHODS:10 volunteers were administered a single dose of Compound amlodipine besylate/atorvastatin calcium tablets(10 mg,p.o.)or multiple doses(10 mg?d-1,p.o.) for 7 days,respectively. Plasma concentrations of amlodipine besylate and atorvastatin calcium were determined by LC-MS/MS and the pharmacokinetic parameters were calculated using DAS software. RESULTS:In single-dose study the pharmacokinetic parameters of amlodipine besylate vs. atorvastatin calcium were as follows:t1/2?(53.4?12.9)h vs.(15.4?4.6)h; Cmax(6.7?1.8)?g?L-1 vs.(18.5?4.4)?g?L-1; AUC0～120 (298.8?97.1)?g?h?L-1 vs. (118.3?48.9)?g?h?L-1; AUC0～∞(412.2?131.5) ?g?h?L-1 vs. (120.0?55.1)?g?h?L-1. In multiple-dose study the pharmacokinetic parameters were as follows:t1/2(49.5?10.3)h vs. (14.4?5.3)h; Cmax(8.7?2.5)?g?L-1 vs. (20.3?5.8)?g?L-1; AUC0～120(451.2?127.1)?g?h?L-1 vs. (136.3?54.9)?g?h?L-1; AUC0～∞(569.3?165.8) ?g?h?L-1 vs. (139.0?61.3)?g?h?L-1; Cav(2.7?0.6)、(8.3?1.3)?g?L-1; R(1.7?0.4) vs. (1.1?0.2). CONCLUSION:The elimination rates of amlodipine besylate and atorvastatin calcium do not change after oral administration of multiple doses of CABACT while slight accumulation of amlodipine besylate is founded.