Objective To prepare a specific anti-lactoferrin single chain variable fragment(ScFv).Methods Anti-lactoferrin clones were screened from a 'naive' phage antibody library against the immobilized lactoferrin antigen,then the clones were transformed to the E.coli HB2151 to give soluble expression of antibody fragments.The culture supernatant containing ScFv was purified by immobilized metal affinity chromatography,and then determined with SDS-PAGE and ELISA.Results The results demonstrated that ScFvs were specific;they did not react with transferrin,lysozyme and bovine serum albumin in ELISA.The SDS-PAGE showed that the ScFvs had high purity through affinity chromatography and the molecular weight of them was about 32 kD.Conclusions The successful generation of the ScFvs against lactoferrin provides a basis for further study and clinical applications for dry eye and other ocular diseases.

AIM: To construct human GPI-B7-1 fusion protein and investigate the therapeutic potentials in the treatment of tumors. METHODS: The chimeric GPI anchored-B7-1 gene was obtained by overlap PCR and inserted into expressing vector pcDNA3.1, named pc3.1/GPI-B7-1. pc3.1/GPI-B7-1was transfected into CHO cells by lipofectamine ~2 000 reagent. The CHO cells, expressing GPI-B7-1 on membranes, were obtained after selecting by G418. That was confirmed by flow cytometry, SDS-PAGE and Western blot. RESULTS: Recombinant vector pc3.1/GPI-B7-1 was successfully constructed and sequence result indicated that it was identical with reference sequence. The protein on transfected CHO cell membrane selected by G418 was confirmed to be GPI-anchored protein by flow cytometry, and GPI-B7-1 approximately 60 kD was conformed by SDS-PAGE and Western blot. CONCLUSION: A large amount of GPI-B7-1 fusion protein was obtained and will be further studied in the treatment of tumors.2? [

AIM: Open reading frame(ORF) of death associa ted protein kinase1(DAPK1) gene was cloned for studying on tumor forming and met astasis.METHODS: Based on nucleotide sequence of DAPK1 gene f rom GenBank, a pair of primers was designed. DAPK1 gene ORF was transfected into Raji cells in expression vector pcDNA3.1(+) with lipofectamine reagent. Morphol ogic assessment of apoptosis was performed with fluorescence microscope cytotoxi city and cell viability was assayed by MTT. RESULTS: DAPK1 gene ORF was amprified from K562 cells by RT-PCR. It was cloned into plasmid pMD18-T and sequenced. There were seven mutation in 4 300 bp nucleotide sequence rel ativel y to DAPK1 nucleotide sequence from GenBank, but six was synonymous mutation and one was single nucleotide polymorphism. 4 300 bp nucleotide of DAPK1 gene O RF was transfected into Raji cells. DAPK1 gene expression was detected in 48 h a fter it was transfected into Raji cells. Then Raji cells showed apoptosis.CONCLUS ION: Large fragment gene was cloned by RT-PCR and transfected into Raji cells successfully. Over-expression of DAPK1 gene induced Raji cells apoptosis.

AIM: To construct pVAX1-GrB. METHODS: Lymphocytes from human laryngeal carcinoma tissue were separated from tumor tissue. The fragment of granzyme B (GrB) was amplified by RT-PCR and was recombined to the downstream of T7 promoter in the vector pVAX1. The construction was transfected into Hep2 cells with lipofectamine 2000. The expression of protein was identified by indirect immunofluorescent antibody assay. RESULTS: It has been proved that the sequence of the RT-PCR product was totally consistent with the data of GenBank by DNA sequencing analysis. The GrB cDNA fragment was cloned into the vector of pVAX1 in the right direction and the open reading fragment of GrB was maintained. The target protein was detected in the transfected Hep2 cells. CONCLUSION: The pVAX1-GrB plasmid was successfully constructed and expressed. [

Objective:To establish the fingerprints of Microctis Folium by ultra high performance liquid chromatography (UPLC); To determine the contents of three flavonoids in the Microctis Folium; To evaluate the quality difference of Microctis Folium from different producing areas. Methods:The fingerprints were performed on Agilent ZORBAX SB C18 column (2.1 mm×150 mm,1.8 μm). The mobile phase was acetonitrile - 0.1 % acetic acid solution with gradient elution at a flow rate of 0.30 ml/min. The column temperature was 30 ℃ and the detection wavelength was 315 nm. The common fingerprint peaks were identified by UPLC-mass spectrometry, and the identification results were confirmed by comparison of reference materials. Waters Cortecs T3 C18 chromatographic column (2.1 mm × 100 mm,1.6 μm) was used for content determination. The mobile phase was methanol-0.1 % formic acid solution with gradient elution at a flow rate of 0.35 ml/min. The column temperature was 30 ℃ and the detection wavelength was 339 nm. The contents of vitexin, isovitexin and narcissoside in 15 batches of Microctis Folium from different habitats were determine. Results:There were 11 common peaks in the fingerprint of Microctis Folium. Identified by mass spectrometry and confirmed by reference substance,10 chemical components were identified, including caffeic acid, p-hydroxycinnamic acid, ferulic acid, vitexin, isovitexin, kaempferol-3-O-rutoside, astragaloside, narcissoside, isorhamnetin-3-O-glucoside and linden glycoside. The similarity between the fingerprints of 15 batches of Microctis Folium and the control fingerprint was greater than 0.95, indicating that the overall similarity of the fingerprints of Microctis Folium from different producing areas was high. The total contents of three active components were 3.23-5.64 mg/g in Yangjiang City, Guangdong, 3.60-5.78 mg/g in Zhanjiang City, Guangdong, 4.68-5.73 mg/g in Yulin City, Guangxi and 3.87-5.21 mg/g in Wuzhishan City, Hainan . There was no significant difference in the content of three active components in different producing areas. Conclusion:The fingerprints and the determination method established in the study are stable and feasible, which can be used for the quality evaluation of Microctis Folium.

Objective:To establish the ultra-high performance liquid chromatography (UPLC) fingerprint and high performance liquid chromatography (HPLC) content determination method of Curcumae Radix standard decoction; To predict the quality markers of Curcumae Radix standard decoction combined with network pharmacology.Methods:UPLC method was used to establish the fingerprint of Curcumae Radix standard decoction, and the common peaks were determined. Combined with chemical pattern recognition techniques such as similarity analysis and clustering analysis, Curcumae Radix standard decoction from different producing areas was studied, and curcumol was used as an index to determine the content of 24 batches of Curcumae Radix standard decoction. At the same time, network pharmacology was used to predict potential of curcumol and (1S, 6β)-1β-Methyl-4-(1-methylethylidene)-7β-(3-oxobutyl) bicyclo [4.1.0] heptan-3-one.Results:A total of 24 batches of Curcumae Radix standard decoction from different habitats were compared and analyzed, and 10 common peaks were calibrated. The similarity of 24 batches of samples ranged from 0.982 to 0.999. Clustering analysis and principal component analysis divided them into three categories. Heat map analysis showed that peak 8 (curcumol) and peak 9 ((1S, 6β)-1β-Methyl-4-(1-methylethylidene)-7β-(3-oxobutyl) bicyclo [4.1.0] heptan-3-one) were the main components. The content of curcumol in 24 batches of Curcumae Radix standard decoction was 0.69-1.87 mg/g; curcumol and (1S, 6β)-1β-Methyl-4-(1-methylethylidene)-7β- (3-oxobutyl) bicyclo [4.1.0] heptan-3-one may regulate the neuroactive ligand-receptor interaction signaling pathway, calcium signaling, and excitation by regulating neuroactive ligand-receptor interaction signaling pathway, calcium signaling, and excitation. It was preliminarily predicted that curcumol and (1S, 6β)-1β-Methyl-4-(1-methylethylidene)-7β-(3-oxobutyl) bicyclo [4.1.0] heptan-3-one were potential quality markers of Curcumae Radix.Conclusion:Curcumol and (1S, 6β)-1β-Methyl-4-(1-methylethylidene)-7β-(3-oxobutyl) bicyclo [4.1.0] heptan-3-one are potential quality markers of Curcumae Radix standard decoction, and the established fingerprint can be used for the quality control of Curcumae Radix standard decoction.

Objective:To establish the HPLC fingerprint of Bolbostemmatis Rhizoma standard decoction; To determine the three effective components with similar structure by quantitative analysis of multi-components by single marker (QAMS); To evaluate the quality of Bolbostemmatis Rhizoma standard decoction.Methods:HPLC was adopted to establish the fingerprints of 15 batches of Bolbostemmatis Rhizoma standard decoction. The Chromatographic column was Waters XBridge Phenyl (4.6 mm×250 mm, 5 μm). The mobile phase was acetonitrile-0.1% phosphoric acid solution with gradient elution. Cluster analysis (HCA) and principal component analysis (PCA) were conducted based on the relative peak area of common peaks. The same method as the fingerprint was used to establish QAMS of tubeimoside A, B, C on Bolbostemmatis Rhizoma standard decoction.Results:There were 14 common peaks in the fingerprint of Bolbostemmatis Rhizoma standard decoction. It was confirmed that the peak 3 was L-tryptophan, the peak 11 was tubeimoside B, the peak 12 was tubeimoside C, and the peak 13 was tubeimoside A. 15 batches of Bolbostemmatis Rhizoma standard decoction from different origins were divided into 3 categories by HCA and PCA. There was no significant difference between QAMS and the external standard method (ESM) through the system suitability inspection. Conclusion:This method is accurate, reliable and has good specificity, which can effectively evaluate the quality of Bolbostemmatis Rhizoma standard decoction.

Objective:To establish the quality evaluation method of Perillae caulis formula granules based on the three kind of quality indexes of standard decoction. Methods:Eighteen batches of Perillae caulis were collected from different habitats according to different technical requirements, eighteen batches of standard decoction and three batches of formula granules were prepared and the paste-forming rates were calculated. The content of Caffeic acid and Rosmarinic acid were determined and calculated by Ultra High Performance Liquid Chromatography (UPLC). Then the fingerprints of standard decoction of and formula granules of Perillae caulis were established by UPLC . The similarity values of fingerprints between formula granules and standard decoction were calculated. Results:The average paste-forming rate of standard decoction was (7.16±1.97)%. The paste-forming rates of three batches of formula granules were 5.52%, 5.25% and 5.34%, respectively. The average content of Caffeic acid and Rosmarinic acid in standard decoction was (12.06±3.37)mg/g. The contents of three batches of formula granules were 5.52, 5.82, 5.77 mg/g, respectively. Seven common fingerprint peaks were identified in the fingerprints of standard decoction and formula granules, three of which were identified as Caffeic acid, N-Feruloyl Octopus amine and Rosmarinic acid by comparison of reference substance. The fingerprints similarity of Perillae caulis dispensing granules and standard decoction were 1.000, 0.995 and 0.997, respectively. Conclusions:The quality indexes of three batches of formulation granules are consistent with standard decoction. This method can provide basis for the establishment of quality standard of Perillae caulis dispensing granules.

Objective:To establish HPLC fingerprints of Aristolochia contorta and honey-processed Aristolochia contorta; To analyze the changes of chemical components before and after honey processing with multivariate statistics; To provide a reference for the study on the toxicity reduction of Aristolochia contorta.Methods:The fingerprints of 11 batches of Aristolochia contorta and honey-processed Aristolochia contorta were established through HPLC. Clustering analysis (HCA), principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and independent sample t-test were used to compare the changes of chemical components of Aristolochia contorta before and after honey processing.Results:The results showed that there were 14 common peaks in the fingerprints of Aristolochia contorta and Aristolochia contorta. 7 common peaks were identified. Both HCA and PCA could clearly distinguish the samples of Aristolochia contorta before and after honey processing. OPLS-DA found and screened 7 differential markers, and the order of difference significance was peak 3 > peak 7 (7-hydroxy aristolochic acid A) > peak 5 (aristolochic acid C)> peak 8 (aristolochic acid D) > peak 6 > peak 2 (Magnolia alkaloid) > peak 14 (aristolochic acid Ⅰ). After honey processing, the content of chemical components represented by peaks 2, 3, 5, 6, 7, 8 and 14 decreased ( P<0.05). Conclusion:This method is simple and specific, which can be used for the fingerprint analysis of Aristolochia contorta and honey-processed Aristolochia contorta, and can effectively distinguish Aristolochia contorta and honey-processed Aristolochia contorta, and provide a reference for the processing research of toxicity reduction of Aristolochia contorta honey processing.

Objective To establish an ultra-high performance liquid chromatography(UPLC)fingerprint and multi-index content determination method of Siraitiae fructus standard decoction.Methods 15 batches of Siraitiae fructus from different producing areas were collected,Siraitiae fructus standard decoction was prepared according to Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules,and the extract rate was calculated.UPLC was used to establish the fingerprint of 15 batches of Siraitiae fructus standard decoction and determine the contents of 11-O-mogroside V,kaempferitrin and mogroside V,which were the main effective components.The chemometrics analysis was used to evaluate the quality of Siraitiae fructus standard decoction and find possible quality markers.Results The extraction rate of 15 batches Siraitiae fructus standard decoction ranged from 24.79％to 34.95％.There were 16 common peaks in the fingerprint,and 4 components were identified.The Siraitiae fructus standard decoction was divided into 2 categories by chemometrics analysis,among which samples from Liuzhou,Guangxi were in one category and samples from Guilin,Guangxi were in another category.Seven differential markers were screened out under the condition of variable importance projection value,and the order was as follows:peak 8＞peak 7＞peak 5＞peak 12(kaempferitrin)＞peak 1＞peak 13＞peak 4.The contents of kaempferitrin,11-O-mogroside V and mogroside V in samples from Guilin,Guangxi were slightly higher than those in samples from Liuzhou,Guangxi.Conclusion The UPLC fingerprint and content determination method established in this study are feasible,which can provide a basis for the quality evaluation of Siraitiae fructus.The results of principal component analysis show that kaempferol is likely to become a quality marker of Siraitiae fructus.