Construction and expression of a human/mouse chimeric CD19 monoclonal antibody: Successful modification of a murine IgM to a chimeric IgG

April 2014
Experimental & Therapeutic Medicine;2014, Vol. 7 Issue 4, p849
Academic Journal
CD19 is a specific surface marker of B cells. A murine IgM-subtype antibody, 2E8, was generated previously and assigned to the CD19 category by the 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens in 1996. In the present study, the 2E8 Fv gene was inserted into a baculovirus shuttle vector and novel protein was expressed in an IgG1 form in the Sf9 insect cell line. VH2E8 and VL2E8 genes were cloned and inserted into the baculovirus shuttle vector pAc-κ-CH3 to form pAc-κ-CH3-VH2E8-VL2E8. Sf9 cells were then transfected with the reconstructed baculovirus shuttle vector. Novel protein expressed by the Sf9 cells was identified by immu-nofluorescence and western blot analysis, while activity levels were analyzed by flow cytometry (FCM). Sequencing demonstrated that the VH2E8 and VL2E8 fragments were inserted into pAc-κ-CH3 correctly. The immunofluorescence, western blot analysis and FCM results indicated that active recombinant antibody was expressed in the cytoplasm of Sf9 cells, but not in the culture supernatant. Thus, functional recombinant antibody was expressed successfully in the cytoplasm of Sf9 cells, but was not secreted into the culture supernatant. Therefore, the present study demonstrates that it is possible to modify mouse IgM to mouse-human chimeric IgG1 while retaining reasonable biological activity.


Related Articles

  • Inhibition of multi-drug resistance of ovarian carcinoma by small interfering RNA targeting to MRP2 gene. Jia-jia Ma; Bi-liang Chen; Xiao-yan Xin; Ma, Jia-jia; Chen, Bi-liang; Xin, Xiao-yan // Archives of Gynecology & Obstetrics;Feb2009, Vol. 279 Issue 2, p149 

    Aims: To investigate the inhibiting mechanism of multi-drug resistance (MDR) using expression vectors of short hairpin RNA (shRNA) in a MDR human ovarian cancer cell line (A2780/cp70).Methods: Two shRNA expression vectors were constructed and introduced into A2780/cp70...

  • Effect of glucose feeding on the glycosylation quality of antibody produced by a human cell line, F2N78, in fed-batch culture. Seo, Jin; Min, Byung; Kim, Yeon; Cho, Jong; Baek, Eric; Cho, Myung; Lee, Gyun // Applied Microbiology & Biotechnology;Apr2014, Vol. 98 Issue 8, p3509 

    The human cell line rF2N78 produces an antibody with a high galactosylation ratio which resembles human IgG. However, it has been observed that the aglycosylated antibody starts to appear when glucose is depleted. To determine whether glucose depletion is a main cause for aglycosylation of the...

  • A simple method to determine IgG light chain to heavy chain polypeptide ratios expressed by CHO cells. Gerster, Anja; Wodarczyk, Claas; Reichenbächer, Britta; Köhler, Janet; Schulze, Andreas; Krause, Felix; Müller, Dethardt // Biotechnology Letters;Dec2016, Vol. 38 Issue 12, p2043 

    Objectives: To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development. Results: Chinese Hamster Ovary (CHO) TurboCell pools containing different...

  • Feasibility of using a dual-promoter recombinant baculovirus vector to coexpress EGFP and GDNF in mammalian cells. JIANZHANG WANG; JUN WANG; CHANGPING CAI; SHILI WANG; SHUAI LIU; SHUO SHI; YIFAN ZHANG; BIAO LI // Experimental & Therapeutic Medicine;2014, Vol. 7 Issue 6, p1549 

    Vectors that are capable of coexpressing two or more exogenous genes for in vitro and in vivo gene delivery are being increasingly studied. The aim of the present study was to explore the feasibility of using the pFastBacâ„¢ Dual vector, under the control of two cytomegalovirus (CMV)...

  • Seroprevalence of Chlamydia pneumoniae Antibodies in a Young Adult Population Sample Living in Verona. Ferrari, M.; Poli, A.; Olivieri, M.; Tardivo, S.; Biasin, C.; Balestreri, F.; dal Molin, G.; Lo Cascio, V.; Campello, C. // Infection;Feb2000, Vol. 28 Issue 1, p38 

    The aim of the study was to evaluate the prevalence of antibodies to Chlamydia pneumoniae in a random population sample of 369 young adults (aged 20-44 years), living in Verona, Italy. lgG and IgM titers were measured by micro-immunofluorescence. lgG antibodies, greater or equal to 16,were found...

  • Application of Western blot analysis for the diagnosis of Encephalitozoon cuniculi infection in rabbits: example of a quantitative approach. Desoubeaux, Guillaume; Pantin, Ana; Peschke, Roman; Joachim, Anja; Cray, Carolyn // Parasitology Research;Feb2017, Vol. 116 Issue 2, p743 

    Diagnosis of Encephalitozoon cuniculi infection in rabbits remains a major veterinary issue. ELISA or immunofluorescence assays are the current reference standards of serological tests. However, these conventional techniques suffer from a lack of accuracy for distinguishing active from past...

  • Baculovirus production for gene therapy: the role of cell density, multiplicity of infection and medium exchange. Carinhas, Nuno; Bernal, Vicente; Yokomizo, Adriana; Carrondo, Manuel; Oliveira, Rui; Alves, Paula // Applied Microbiology & Biotechnology;Jan2009, Vol. 81 Issue 6, p1041 

    One of the major concerns regarding the use of insect cells and baculovirus expression vectors for the production of recombinant proteins is the drop in production observed when infecting cultures at high cell densities; this work attempts to understand this so-called cell density effect in the...

  • Transient Receptor Potential Channel TRPM8 Agonists Stimulate Calcium Influx and Neurotensin Secretion in Neuroendocrine Tumor Cells. Mergler, Stefan; Strowski, Mathias Z.; Kaiser, Simone; Plath, Thomas; Giesecke, Yvonne; Neumann, Marleen; Hosokawa, Hiroshi; Kobayashi, Shigeo; Langrehr, Jan; Neuhaus, Peter; Plöckinger, Ursula; Wiedenmann, Bertram; Grötzinger, Carsten // Neuroendocrinology;Feb2007, Vol. 85 Issue 2, p81 

    TRPM8 is a member of the melastatin-type transient receptor potential ion channel family. Activation by cold or by agonists (menthol, icilin) induces a transient rise in intracellular free calcium concentration ([Ca2+]i). Our previous study demonstrated that Ca2+-permeable cation channels play a...

  • Chikungunya Fever in Travelers Returning to Europe from the Indian Ocean Region, 2006. Panning, Marcus; Grywna, Klaus; Van Esbroeck, Marjan; Emmerich, Petra; Drosten, Christian // Emerging Infectious Diseases;Mar2008, Vol. 14 Issue 3, p416 

    Chikungunya fever has spread through several Indian Ocean islands and India, including popular travel destinations. To compare usefulness of diagnostic tests and to understand reasons for the magnitude and severity of an outbreak, we used 3 diagnostic methods to test 720 samples from 680...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics