ABSTRACT
Objective To investigate the application effect of the Beckman Coulter PK 7300 automatic blood group analyzer and the microplate method in ABO blood group screening .Methods A total of 12000 EDTA anticoagulation whole blood samples from January to May 2015 were collected from voluntary blood donors .The Beckman Coulter Pk7300 automatic blood group analyzer and STAR sampling microplate manual colorimetric method were to conduct the detection analysis .Results The accuracy for detecting ABO blood group had no statistically significant difference between the two methods (P>0 .05) .In the ABO blood group screening , the detection rates of ABO subtype and antibody weakening in the Beckman Coulter Pk 7300 automatic blood group analyzer were higher than those in the micro plate method .3 cases of ABO blood group typing and reverse typing were consistent in the detection by the Beckman Coulter Pk7300 automatic blood group analyzer ,but was inconsistent in the detection by the microplate method .4 cases of ABO blood group typing and reverse typing were inconsistent in the the detection by the Beckman Coulter Pk 7300 automat-ic blood group analyzer ,but was consistent in the detection by the microplate method .Conclusion The Beckman Coulter Pk7300 automatic blood group analyzer can safely and effectively conduct the ABO blood group screening in blood donors .The samples of suspicious detection results still need to conduct the manual interpretation by combining with the test tube method .
ABSTRACT
In recent years, Chinese hamster ovary (CHO) production vessel volume has reached more than 1 000 L in Chinese biopharms, and 10 000 L in foreign big biopharms, such as Lonza and Genetech. In general, there are some steps seed bioreactor for seed expansion, which decreases the efficiency of production process. In this work, a perfusion-based process was developed to drastically increase the split ratio during the scale-up of CHO cell cultures. Fed-batch cultures were inoculated with cells propagated in either batch or perfusion cultures that grown in disposable Cellbags using the WAVE Bioreactor system. The higher cell concentration of 2 x 10(7) cells/mL with 95% viability allowed to increase the split ratio to about 1:50-1:100 for inoculum propagated in perfusion culture. The method described here could reduce the number of required expansion steps and eliminate two or three bioreactors. Disposable perfusion bioreactor with only a few liters working volume have the potential to directly inoculate volumes of up to 1 000 liters. This would allow to shorten process time in these bioreactors, which often are the bottleneck in plant throughput.
Subject(s)
Animals , Cricetinae , Bioreactors , CHO Cells , Cell Biology , Cell Culture Techniques , Methods , CricetulusABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of the 25 kD hepatitis B e antigen (HBeAg) precursor that only exist inside hepatocytes and study its effect on the pathopoiesis of hepatitis B and QIAGEN expression and purification system.</p><p><b>METHODS</b>Hepatitis B virus (HBV) preC/C gene for the 25 kD HBeAg precursor was cloned into the expression vector pQE30 and the 25 kD HBeAg precursor was expressed in Escherichia coli (E. coli) and purified. Its antigenicity for 21 kD mature HBeAg was tested by western blot analysis.</p><p><b>RESULTS</b>Cloned fragments in the expression vector were sequenced and verified to be homogeneous with that of HBV (ayw subtype). Expression of the HBeAg precursor in E. coli under the transcriptional regulation of T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 25 kD. Recombinant HBeAg precursor exhibited identical potencies with 21 kD mature HBeAg that reacted with anti-HBeAg antibodies. The purification rate of the expressed HBeAg precursor was up to 89.6% and the yield of purified HBeAg precursor from this procedure was 2.4 mg/L.</p><p><b>CONCLUSION</b>25 kD HBeAg precursor exhibited biological activity and might play an important role in pathopoiesis of hepatitis B.</p>
Subject(s)
Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetics , Gene Expression , Hepatitis B e Antigens , Genetics , Metabolism , Plasmids , Genetics , Protein Precursors , Genetics , Metabolism , Recombinant Proteins , MetabolismABSTRACT
To study the characteristics of EBV transformed human peripheral blood B cell lines from hepatitis B patients and to provide a basis for further studies on the cellular immune function of hepatitis B patients.High titer of EBV was produced by B95 8 cell by induction with sodium n butyrate,and peripheral mononuclear cells from two different groups of hepatitis B patients were harvested and infected with EBV. The results showed that 4～8 weeks after infection 19 EBV transformed B cell lines from hepatitis B patients were established. It was found that colony formation of cells from A group appeared 2～3 weeks later than B group. Cells grew healthily and had good activity after 4 weeks of freezing.CD19 and CD20 were detected in 88 34% and 37 48% of the immortalized B cell membrane respectively. HBV DNA could not be detected in a11 19 immortalized B cell lines.It suggested that the time needed for the establishment of B cell lines was related with the immune state of the patients.HBV DNA could not exist persistently, and it would disappear finally in the immortalized B cell lines.
ABSTRACT
Objective To study the biological significance of the common mutant preC/C gene in clinical HBV in China. Methods Site directed mutagenesis based on the unique enzyme site elimination was used to construct eukaryocyte expression vector with mutant HBV/C gene(V60、G87、L97). Expression vectors with wild and mutant preC/C gene were transferred into HepG2 cell. Culture supernatant was detected by ELISA for HBeAg. Results Result of DNA sequencing showed that the constructed mutant HBV preC/C gene had only one specific site variation compared with the wild type sequence. Goal DNA fragment was detected positive in the HepG2 cells transferred with wild and mutant preC/C gene. A value of HBeAg in the supernatant of the cells harboring L97 variant was higher than that of the wild and other variant strains( P
ABSTRACT
Objective In order to study the biological significance of HBV/C gene variant in vitro.Methods Retroviral vector pXT1 was used to construct the HBV/C gene expression vector and the recombinant plasmid pXT1-HBV/C was used to transfect immortalized human peripheral blood B cell lines to express HBcAg steadily in the host cells.Results plasmid pXT1-HBV/C was detected positive by PCR as well as enzyme digestion with Bgl Ⅱ and Xho Ⅰ.Meanwhile.transfected cells was detected to contain HBV/C gene by PCR and HBcAg was expressed in 47.4%of the ceils by means of flow cytometry.Conclusion Retroviral expression vector with HBV/C gene can transfeet into eucaryotic cells effectively and express the goal gene steadily.The recombinant cells may be used in the systematic studies on the biological significance of HBV/C gene variant.