2.Clinical Significance of Detection Antibody of Liver Antigens in Patients with Autoimmune Hepatic Diseases and Viral Hepatitis
Jiang LIN ; Ke WANG ; Sunyun CHEN
Journal of Modern Laboratory Medicine 2015;(3):153-156
Objective To study the positive incidence and clinical significance of auto-antibody of liver antigens in the serum of patients with autoimmune hepatitis diseases and viral hepatitis.Methods The serum samples with hepatic diseases were col-lected in Jiangyin People’s Hospital from 2010 to 2014 year.Patients were divided into three groups according to diseases:autoimmune hepatic disease group including autoimmune hepatitis (AIH)12 cases and primary biliary cirrhosis (PBC)36 case;viral hepatic group including HAV 23 cases,HBV 30 cases,HCV 14 cases and HEV 8 cases;normal control group 30 cases.Auto-antibody of live antigens AMA-M2,LKM-1,LC-1,SLA/LP,GP210 and SP100 were tested respectively by west-ern blotting assay.Results The positive rates of anti-AMA-M2,anti-LKM-1,anti-LC-1,anti-SLA/LP,anti-GP210,anti-SP100 were 16.7%,16.7%,8.3%,25%,0% and 16% respectively in AIH group,83.3%,0%,0%,0%,44.4% and 27.8% respectively in PBC group.Patients with viral hepatitis,one case were anti-AMA-M2 positive,one case were anti-GP210 positive and three case with positive anti-SP100.anti-AMA-M2,anti-GP210 and anti-SP100 were detected more fre-quently in autoimmune liver diseases group than viral hepatitis group (χ2 = 33.9,10.6 and 8.8,P < 0.05),anti-SLA/LP were detected more frequently in AIH group than PBC group (χ2 = 6.4,P <0.05),while anti-AMA-M2,anti-GP210 and were detected more frequently in PBC group than AIH group (χ2 =15.1 and 6.1,P <0.05).The positive rate of viral hepat-ic group was a very low frequency,there are little statistically significant difference (χ2 =1.1,P >0.05)compared with nor-mal control group.Conclusion The antibody of liver antigens are useful clinically for diagnosing and classifying autoimmune liver disease.
3.Researches of Effects of Phthalate Esters on Rodents and Human
Xiaoyi WANG ; Xingtao LIN ; Huiming KE
Journal of Environment and Health 2007;0(09):-
Phthalate esters have been used as the plasticizers for about 80 years. As a kind of environmental hormone and universal pollutants, they are found widely in air dust, industrial waste water, river, soil and solid waste, moreover, they have been detected in food, drinking water and body fluid. In this paper, the new progresses of the effects of phthalate esters on the rodents and human, including reproductive toxicity and liver toxicity, were summarized and the short-term, quick and accumulative actions of phthalates mono-esters, metabolite of phthalate esters, on the rodents and human body were also reviewed.
4.SCREENING AND CLONING OF THE GENES OF PROTEIN INTERACTING WITH THE NONSTRUCTURAL PROTEIN 5A OF HEPATITIS C VIRUS
Medical Journal of Chinese People's Liberation Army 1982;0(01):-
Protein protein binding is the basis of virus and host cell interactions. With the application of technology of studying of protein interactions, more knowledge of replication and pathogenesis of hepatitis C virus (HCV) could be acquired. Non structure protein NS5A is one of the important regulatory factors in virus replication , transcription and signal transduction, but there are controversy in effect on HCV pathogenesis and resistance to interferon ?(IFN?). In order to describe the relationship between NS5A and host proteins, we use yeast two hybrid system 3 to screen the gene encoding proteins that could interact with NS5A from human hepatocyte library. Thirty five clones were obtained including apo A1, apo A2, apo B100, haplotype mitochondrion complete genome, phosphatidic acid phosphatase type 2B, albumin similar to tumor endothelial marker 5 precursor, matrix metalloproteinase 14, and three of the Homo sapiens hypothetical proteins. The study paved a way for further studies on the pathogenesis of HCV NS5A
5.SCREENING AND CLONING OF GENE OF HEPATOCYTE PROTEIN INTERACTING WITH HCV NS3 PROTEIN
Medical Journal of Chinese People's Liberation Army 1982;0(01):-
To clone the genes of hepatocyte protein interacting with hepatitis C virus NS3 protein, "bait" plasmids of hepatitis C virus NS3 were constructed. After verifying that hepatitis C virus NS3 protein could be steadily expressed in AH109 yeast strain, yeast two hybrid assay was berformed by mating AH109 with Y187 that pre transformed with liver cDNA library plasmids pACT2, and the diploidy yeast cells were plated on quadruple dropout (QDO) medium and assayed for X ? gal activity. Nineteen yeast colonies that could grow on QDO and had ? gal activity were obtained, then the library plasmids were extracted and sequenced. The gene sequences from the 19 positive colonies were aligned with the genes deposited in GenBank. It was found hepatitis C virus NS3 protein could interact with some proteins which have different functions.
6.CLONING OF THE HEPATITIS C VIRUS NONSTRUCTURAL PROTEIN 5A BINDING PROTEIN 37 CODING GENE
Medical Journal of Chinese People's Liberation Army 2001;0(09):-
The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) has been shown to interact with a variety of cellular proteins and implicated in the regulation of cell growth, interferon resistance, and other cellular signaling pathways. Using the yeast-two hybrid method, we have isolated a clone that encodes a novel NS5A--associated binding protein: NS5ABP37. Reverse transcription polymerase chain reaction (RT-PCR) method was employed to amplify the full fragment,and the plasmid pGADT7-NS5ABP37 with the Saccharomyces cerevisiae vector pGADT7 was constructed. To prove the interaction, yeast cell Y187 transformed with pGADT7-NS5ABP37 was mated with yeast cell AH109 containing pGBKT7-NS5A to verify the interaction between the novel protein coded by the new gene NS5ABP37 and NS5A.
7.Cloning and expression of NS3 gene of hepatitis C virus in yeast
Chinese Journal of Infectious Diseases 1999;0(01):-
Objective HCV NS3 protein plays an important role in disease caused by HCV. We investigate the gene expression of HCV NS3 in yeast for future study of the function of the protein. Methods PCR was performed to amplify the gene of HCV NS3 from the plasmid pBRTM/HCV containing the whole fragment of HCV and the gene was cloned into pGEM T vector. Thereafter, HCV NS3 gene was cut from pGEM T vector and cloned into yeast expression plasmid pGBKT7, and recombinant pGBKT7∶NS3 was transformed into yeast AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting. Results HCV NS3 gene was successfully cloned into pGBKT7. The results of SDS PAGE and Western blotting assay showed that the molecular weight of the expressed product was about 22000 Da and HCV NS3 protein was existed within yeast cells.Conclusions HCV NS3 was successfully expressed in yeast expression system.
8.Affirming marker peaks for fingerprint chromatography of Shengmai Injection
Jing XIA ; Ke WANG ; Gangli WANG ; Shen JI ; Ruichao LIN
Chinese Traditional Patent Medicine 1992;0(03):-
AIM: To affirm marker peaks for the fingerprint chromatography of Shengmai Injection. METHODS: LC-MS/MS method was used, with a Waters symmetryshield TM RP_ 18 column(4.6 mm?250 mm; 5.0 ?m), acetonitrile-water as a mobile phase, The detection wavelength was at 203 nm. Ion trap mass spectrum. RESULTS: Affirming marker peaks for fingerprint chromatography of Shengmai Injection and 10 marker peaks were affirmed. CONCLUSION: The method can affirm marker peaks for the fingerprint chromatography of Shengmai Injection. It is simple, accurate, and has practicality.
9.Expression and purification of Trop2 virus like particles
Lin WANG ; Xi WANG ; Ke DONG ; Huizhong ZHANG
International Journal of Laboratory Medicine 2016;37(8):1020-1022,1025
Objective To gain the virus like particles (VLPs) based on Trop2 targets ,which provided the basis for further in vi‐vo induced experiments and anti‐tumor vaccine studies .Methods Using molecular cloning method to constract eukaryotic expres‐sion vector of pCAGGs/Trop2 and baculovirus expression vector of pFastbac1/Trop2 ,expression product of pCAGGs/Trop2 in HeLa cells were intended to be to be anchored to the cell membrane by the methods of Immunohistochemistry ;pFastbac1/Trop2 were transformed into E .coliDH10bac isolates to gain recombinant bacmids ,which were transfected into insect cells to express re‐combinant baculovirus with Gag rBV ,then sucrose density gradient centrifugation ,Western Blot and electron microscope were per‐formed to purify and identify the Trop2 VLPs .Results Building recombinant bacmids which were transfected into insect cells to express recombinant baculovirus with Gag rBV ,gained the recombinant Virus like Particles .Conclusion Trop2 VLPs was success‐fully prepared ,which laid the foundation for the subsequent induction of humoral and cellular immune response .