Construction,expression of I-Ad/IgG2b Fc dimer fusion protein and its identification / 中国免疫学杂志

Objective:To construct I-Ad/IgG2b Fc baculovirus expression vector and express I-Ad/IgG2b Fc dimer fusion protein in Sf9 insect cells.Methods:I-Ad α,I-Ad β and IgG2b Fc gene sequences were amplified from BALB/c mouse lymphocytes by RT-PCR.I-Ad α and I-Ad β were connected with the leucine zipper sequence Fos and Jun respectively by overlapping PCR to form I-Ad α-Fos and I-Ad β-Jun.I-Ad α-Fos and IgG2b Fc fragments were ligated by restriction sites Xba I to form I-Ad α-Fos-IgG2b Fc recombination sequence.I-Ad α-Fos-IgG2b Fc and I-Ad β-Jun fragments were inserted to PPH and PP10,which were the downstream of the promoters in the plasmid pFastBacTMDual,to form pFastBacTMDual+[I-Ad/IgG2b Fc] recombinant plasmids.The constructed vector was identified by PCR,restriction endonuclease and sequencing.The recombinant plasmids pFastBacTMDual+[I-Ad/IgG2b Fc] was transferred into the DH10Bac competent cell to form recombinant baculovirus Bacmid+[I-Ad/IgG2b Fc].The recombinant baculovirus was transfected into Sf9 insect cells by liposome transfection reagent.After infected with Sf9 insect cells,the supernatant was collected and concentrated by PEG20000 to obtain I-Ad/IgG2b Fc dimer fusion protein.The fusion protein was detected by double-antibody sandwich ELISA and Western blot.Results:PCR,restriction enzyme digestion and sequencing confirmed that the recombinant vector pFastBacTMDual+[I-Ad/IgG2b Fc] had the correct sequence.The double antibody sandwich ELISA and Western blot showed that recombinant bacmid could successfully infect Sf9 insect cells,and the expressed fusion protein had the correct conformation.Conclusion:The pFastBacTMDual+[I-Ad/IgG2b Fc] baculovirus expression vector was successfully constructed and expressed in Sf9 insect cells,laying a foundation for the study of I-Ad-restricted T cells.

THE DEVELOPMENT OF THE CYTOARCHITECTURE OF THE HUMAN FETAL VISUAL CORTEX (AREA 17) PRIOR TO THE APPEARANCE OF SIX LAMINAE OF BRODMANN——A SCANNING ELECTRON MICROSCOPIC (SEM) OBSERVATION / 解剖学报

Tissue was obtained from 14 aborted human fetuses, ranging from 13-32 weeks of gestation (wg). The crown-rump length (CR) ranged from 8.3-33 cm. Frontal sections of the specimens were prosessed for SEM and observation were focused on the areas adjacent to the middle part of the calcarine fissure.At 13 wg (CR 8.3 cm), the visual cortex (area 17) was composed of five zones: viz., the ventricular zone, the subventricular zone, the intermediate zone, the cortical plate and the marginal zone. These five zones showed a series of transformations with increasing age. 1) The ventricular zone became progressively thinner, mitotic activity of the ventrieular cells decreased progressively and finally the ventricular ceils differentiated into a single layer of ependymal cells. 2) The subventricular zone and the inter mediated zone were replaced by fiber bundles of white matter. 3) The cortical plate increased in width, exhibited the greatest growth rate, and became differentiated. At 21 wg (CR 20cm), the lower part of the cortical plate first gave rise to laminae VI and V. At 23 wg (CR 22cm), lamina Ⅳ was established in the middle part of cortical plate. At 26 wg (CR 25cm), laminae Ⅲ and Ⅱ could be identified in the upper part of cortical plate. 4) The marginal zone transformed into lamina Ⅰ at its original site.