1.Immuno-screening of Schistosoma japonicum cercariae cDNA library by the sera of anti-soluble cercariae 66 to approximately 68 kD antigens.
Yong-Hua QIN ; Shuai-Feng ZHOU ; Shi-Ping WANG
Journal of Central South University(Medical Sciences) 2008;33(12):1076-1081
OBJECTIVE:
To obtain the coding genes related to Schistosoma japonicum (Sj) cercariae 66 to approximately 68 kD antigens,and to provide antigens for diagnosis and vaccine of schistosomiasis.
METHODS:
Sj cercariae cDNA library was screened using the monospecific anti-sera of rabbit against soluble cercariae 66 to approximately 68 kD antigens as probes.The inserted cDNA fragments of the positive clones were amplified with PCR and identified by agarose gel electrophoresis. Four strong positive clones were further sequenced and analyzed through the internet NCBI/BLAST software.
RESULTS:
Twenty-one positive clones were obtained, 10 of which revealed a single band (0.5 to approximately 3.0 kb).The 4 strong positive clones showed high identity to SJCHGC05187,SJCHGC05173,SJCHGC06989, and SJCHGC01894 at the nucleotide level.
CONCLUSION
Four coding genes related with Sj antigens are obtained.
Animals
;
Antibodies, Helminth
;
immunology
;
Antigens, Helminth
;
immunology
;
Cercaria
;
genetics
;
immunology
;
DNA, Complementary
;
genetics
;
Gene Library
;
Immune Sera
;
immunology
;
Schistosoma japonicum
;
genetics
;
immunology
2.Emergency prophylactic effects of the avian influenza virus immunized serum on the infected mice.
Cheng-yu WANG ; Hua-lei WANG ; Na FENG ; Song-tao YANG ; Yu-wei GAO ; Tei-cheng WANG ; Xiao-huan ZOU ; Xian-zhu XIA
Chinese Journal of Preventive Medicine 2008;42(11):814-817
OBJECTIVETo evaluate emergency prophylactic effects of the avian influenza virus immunized serum on experimentally infected mice.
METHODSSerum HI antibody titers of 30 mice were detected at day 1 to 19 after being inoculated with 0.2 ml immune serum to estimate half life of immune serum. Ten mice clinical symptom was recorded to estimate the serum security after mice injected 1.5 ml immune serum. Seventy mice were randomly divided into 7 groups according to random number table and inoculated with 0.2 ml, 0.1 ml and 0.05 ml immune serum respectively via intraperitoneal injection on day 8, 4 and 1 prior to challenged with 10 LD(50) influenza virus intranasal. Mice were observed continually for 14 days to calculate the morbidity, mortality, average survival days and compare the lung index and viral titers in lung.
RESULTSSerum HI antibody titers of mice which inoculated with 0.2 ml immune serum maintained 2(6) in 15 days after injection, but drawdown after day 17, the mice injected 1.5 ml immune serum were all alive and none onset. The survival rate of mice which injected 0.2 ml serum on the day 8, 4, 1 before challenge was 80%, 100% and 100%, and the average survival period was 13.1 days, 14.0 days and 14.0 days respectively. The survival rate of mice which injected 0.1 ml and 0.05 ml serum on day 1 before challenge was 100% and 50%, and the average survival days were 14.0 days and 11.7 days respectively. The mice lung index of experimental groups (0.0096 +/- 0.0033 - 0.0145 +/- 0.0060) was smaller than that of viral control group (0.0199 +/- 0.0025), with a statistical significance (P value 0.0022 - 0.0470, < 0.05). The viral titers in lung were significantly decreased by 2 titer as compared to the viral controls.
CONCLUSIONThe avian influenza virus immunized serum might contain the emergency prophylactic effects and could be developed as an agent for possible human-avian influenza pandemic.
Animals ; Antibodies, Viral ; immunology ; Immune Sera ; immunology ; Immunization ; Influenza A Virus, H5N1 Subtype ; immunology ; Male ; Mice ; Mice, Inbred Strains ; Orthomyxoviridae Infections ; immunology
3.Immunochemical Characterization of Brain and Pineal Tryptophan Hydroxylase.
Young In CHUNG ; Dong Hwa PARK ; Myoungsoon KIM ; Harriet BAKER ; Tong Hyup JOH
Journal of Korean Medical Science 2001;16(4):489-497
Recombinant mouse tryptophan hydroxylase (TPH) was expressed in Escheri-chia coli, using a bacterial expression vector and has been purified to homogeneity by sonication followed by Sepharose 4B column chromatography and native slab gel electrophoresis. This purified enzymatically active TPH protein was used for production of a specific antiserum. This antiserum identified the predicted TPH band (molecular weight, 54 kDa) on Western blot of crude extracts from the rat and mouse dorsal raphe, and the rat pineal gland. However, this antiserum recognized an additional protein band of lower molecular weight (48 kDa) in pineal extract. It is not clear whether the 48 kDa TPH band represents an isozyme or a protease cleavage product of TPH. Since the pineal gland contains higher TPH mRNA and lower TPH activity when it is compared with dorsal raphe nucleus enzyme, this lower molecular weight TPH may participate in the reduced TPH specific activity. In addition, there are no specific TPH inhibitors in the pineal gland and this lower molecular weight TPH is inactive or has a very low specific activity. This antiserum specifically immunostained serotonergic cell bodies in the dorsal raphe nuclei, some large caliber serotonergic processes in the dorsal raphe area as well as terminals in the olfactory bulb. It also immunolabeled the pineal gland and immunoprecipitated equally well TPH protein from the dorsal raphe nucleus and the pineal gland in a concentration-dependent manner.
Animal
;
Blotting, Western
;
Brain/*enzymology
;
Cross Reactions
;
Electrophoresis
;
Immune Sera/immunology
;
Immunohistochemistry
;
Mice
;
Pineal Body/*enzymology
;
Rabbits
;
Rats
5.Prokaryotic expression of Staphylococcus aureus Clumping factor B and evaluation of the antiserum-mediated opsonic activity.
Lili ZHANG ; Shanshan CAO ; Yifeng ZHANG ; Chunling XUAN ; Youqiang WU ; Songhua HU
Chinese Journal of Biotechnology 2015;31(11):1623-1631
Staphylococcus aureus is a major cause of hospital-acquired infection. Because the bacteria are very easy to become resistant to antibiotics, vaccination is a main method against S. aureus infection. Clumping factor B (ClfB) is an adhesion molecule essential for S. aureus to colonize in the host mucosa and is regarded as an important target antigen. In this study, we successfully used Escherichia coli to express a segment encoding the N1-N3 regions of ClfB protein (Truncated-ClfB) cloned from S. aureus. The protein was purified by affinity and ion exchange chromatographies and gel filtration. Rabbits were immunized three times with purified Truncated-ClfB. After that, blood was collected to prepare serum which were then used for measurement of antibody level. Phagocytosis of S. aureus opsonized by the serum was determined by a flow cytometry. Results show that the serum IgG titer reached 1:640 000. Phagocytosed S. aureus by polymorphonuclear leukocytes were significantly more when the bacteria were opsonized by the serum from Truncated-ClfB immunized rabbits than those from no immunized group (P < 0.01). Therefore, the results indicated that Truncated-ClfB could be a promising vaccine candidate against S. aureus infection.
Adhesins, Bacterial
;
immunology
;
Animals
;
Antibodies, Bacterial
;
blood
;
Escherichia coli
;
Flow Cytometry
;
Immune Sera
;
Immunoglobulin G
;
blood
;
Opsonin Proteins
;
immunology
;
Phagocytosis
;
Rabbits
;
Staphylococcal Infections
;
immunology
;
Staphylococcus aureus
6.Preparation and identification of polyclonal antiserum against angiotensinogen.
Qing-Jun MENG ; Jian LI ; Wen-Hong ZHANG ; Le-Ning ZHANG ; Li-Fan ZHANG
Acta Physiologica Sinica 2003;55(1):110-113
For studying the expression and distribution of angiotensinogen (AGT), the C-teminus of rat AGT gene was expressed in E.coli. Rabbits were immunized with expressed AGT protein and sera from different rabbits were raised. ELISA showed a high titre (1:25600) of the antiserum. With the antiserum, Western blotting recognized not only the prokaryotic expressed AGT, but also the endogenous AGT protein in liver tissue of both rats and humans. Using this antiserum, immunohistochemistry showed the expression of AGT protein in islet cells of human pancreas as well as in epithelium of human bile duct. These results suggest that the prokaryotic expressed AGT protein is an effective immunogen for the preparation of anti-AGT antiserum. Our present work provides an important tool for study of the pathophysiological role of AGT as well as local renin-angiotensin system.
Angiotensinogen
;
genetics
;
immunology
;
Animals
;
Antibodies, Monoclonal
;
biosynthesis
;
Escherichia coli
;
genetics
;
metabolism
;
Humans
;
Immune Sera
;
biosynthesis
;
immunology
;
Immunization
;
Rabbits
;
Rats
;
Recombinant Proteins
;
biosynthesis
;
genetics
;
immunology
;
Renin-Angiotensin System
;
physiology
7.Expression of human HZF1 in E. coli and preparation of antibody against human HZF1 protein.
Han PENG ; Xin ZHANG ; Zhan-Wen DU ; Jun-Wu ZHANG
Acta Academiae Medicinae Sinicae 2007;29(6):772-776
OBJECTIVETo express human HZF1 fusion protein in E. coli and to obtain an anti-HZF1 antibody.
METHODSA DNA fragment encoding non-zinc finger region of HZF1 protein was inserted into pET30a vector to get the recombination expression plasmid pET30a-HZF1. E. coli was transformed with pET30a-HZF1 and the selected clones were cultured with isopropy-beta-D-thiogalactoside induction. The proteins were prepared from the culture and the fusion protein was purified by Ni column. Rabbits were immunized and reinforced three times with the purified fusion protein. The antiserum was collected and the titer and the specificity of the antibody were checked by ELISA and Western blot.
RESULTSAntibody against HZF1 was obtained and its titer was more than 1:100 000, as proven by ELISA. Western blot analysis showed specific reaction between this antibody and HZF1 fusion protein or the endogenetic HZF1 protein in hemin-induced K562 cells.
CONCLUSIONSThe specific antibody against HZF1 is obtained. The antibody may have potential application in farther HZF1 function study and HZF1 determination in tissues and cells.
DNA-Binding Proteins ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Transfer Techniques ; Immune Sera ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Transformation, Bacterial
8.Preparation and characterization of follwing the national standard anti-Brucella abortus serum, bovine.
Cui LI ; Fushi GUAN ; Zhihong DAI ; Hui JIANG ; Fang WEN ; Lianshou LU ; Zaishi WANG
Chinese Journal of Biotechnology 2011;27(5):812-816
To prepare anti-Brucella abortus serum used for calibrate the agglutination test follwing the national standard, 4 anti-Brucella abortus sera were obtained from 4 cows infected with Brucella abortus naturally. By potency testing, the third serum was selected. Sterility, vaccum degree, residual moisture, uniformity and stability of this standard material were tested and proved to meet the national standard. Referring to the international standard, RBT (Rose-Bengal plate agglutination test), SAT (standard tube agglutination) and CFT (complement fixation test) titers of this standard material were measured to be 1:160 "+" 1:2 400 "++" and 1:800 "++", which are identical with the collaborative assay results. International unit of the standard material is 4 000 IU/mL.
Agglutination Tests
;
veterinary
;
Animals
;
Antibodies, Bacterial
;
blood
;
Antigens, Bacterial
;
immunology
;
Brucella abortus
;
immunology
;
Brucellosis, Bovine
;
diagnosis
;
Cattle
;
China
;
Complement Fixation Tests
;
veterinary
;
Immune Sera
;
Reference Standards
9.Synthesis and identification of antigenic conjugates of podophyllotoxin.
Dun-Ming XU ; Xiang-Yang YU ; Ying-Qian LIU ; Jun-Tao FENG ; Jung-Qing MA ; Xian-Jin LIU ; Xing ZHANG
Acta Pharmaceutica Sinica 2005;40(6):518-524
AIMTo synthesize and identify artificial antigen of podophyllotoxin for the production of podophyllotoxin polyclonal antibody.
METHODSThe hapten was synthesized by two different chemical approaches and characterized by TLC, IR, NMR, and MS. Mixed anhydride reaction (MAR) and active ester method (AEM) were used to couple the podophyllotoxin to carrier proteins (BSA and OVA). Characterization of artificial antigens was done by using spectroscopy and electrophoresis. The anti-podophyllotoxin polyclonal antibodies were obtained through immunizing rabbits.
RESULTSThe results from IR, NMR and MS showed that 4-O-succinoyl podophyllotoxin (hapten) was successfully synthesized. The coupling molar ratios of the hapten and carrier proteins were 88.6 for Hapten-BSA1, 40.3 for Hapten-BSA2, 17.8 for Hapten-OVA1, and 54.2 for Hapten-OVA2. Hapten conjugates coupled with BSA yielded two sets of the specific and affinitive polyclonal antibodies. One set of antibodies showed an IC50 value of 2.21 microg.mL(-1) with a detection limit of 0.12 microg.mL(-1).
CONCLUSIONAntigenic conjugates were artificially synthesized, and based on these artificial antigens, polyclonal antibodies against podophyllotoxin were raised from rabbits immunized with two different immunogens and characterized with an indirect ELISA format.
Animals ; Antibodies ; analysis ; Antibody Affinity ; Antibody Formation ; Antineoplastic Agents, Phytogenic ; immunology ; Enzyme-Linked Immunosorbent Assay ; Haptens ; chemistry ; immunology ; Immune Sera ; chemistry ; Male ; Ovalbumin ; immunology ; Podophyllotoxin ; immunology ; Proteins ; immunology ; Rabbits ; Serum Albumin, Bovine ; immunology
10.Immune Protection against H9N2 Provided by H1N1 Pre-infection in Pigs.
Jia WANG ; Maocai WU ; Wenshan HONG ; Zuoyi ZHENG ; Rirong CHEN
Chinese Journal of Virology 2015;31(4):357-362
To explore the impact of the history of infection by the influenza A virus subtype H1N1 on secondary infection by the influenza A virus subtype H9N2, pigs non-infected and pre-infected with H1N1 were inoculated with H9N2 in parallel to compare nasal shedding and seroconversion patterns. Unlike pigs without a background of H1N1 infection, nasal shedding was not detected in pigs pre-infected with H1N1. Both groups generated antibodies against H9N2. However, levels of H1N1 antibodies in pigs pre-infected with H1N1 increased quickly and dramatically after challenge with H9N2. Cross-reaction was not observed between H1N1 antibodies and H9N2 viruses. These findings suggest that circulation of the H1N1 virus might be a barrier to the introduction and transmission of the avian H9N2 virus, thereby delaying its adaptation in pigs.
Animals
;
Antibodies, Viral
;
immunology
;
Cross Reactions
;
Immune Sera
;
immunology
;
Influenza A Virus, H1N1 Subtype
;
immunology
;
physiology
;
Influenza A Virus, H9N2 Subtype
;
immunology
;
Orthomyxoviridae Infections
;
blood
;
immunology
;
Species Specificity
;
Swine
;
immunology
;
virology