OBJECTIVETo construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique.

METHODSTotal RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry.

RESULTSUsing 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)].

CONCLUSIONUsing 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

Animals ; Antibodies, Viral ; CHO Cells ; Cell Surface Display Techniques ; Cricetinae ; Cricetulus ; Dengue Virus ; immunology ; Gene Library ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; immunology ; Transfection

Objective:To investigate the difference of functional connectivity(FC) between the habenula and other brain regions in the patients with first-episode current depression (fMDD) and remitted depression (rMDD).Methods:Thirty-five patients with first-episode current depression (fMDD), 35 patients with remitted depression (rMDD) and 30 healthy controls (HC) matched with gender, age and education years were scanned by resting-state fMRI. Hamilton Depression Scale (HAMD 17) was used to assess the severity of the patients. After preprocessing, seed-based FC analysis was performed on the habenula. Pearson correlation analysis was performed between the FC values and HAMD 17 and duration of disease. Results:(1) Compared with the HC group, FC decreased between the habenula and left middle occipital gyrus(l-MOG) in fMDD group (x, y, z=-48, -84, 3, t=-4.335, P<0.05), while FC increased between the habenula and left supramarginal gyrus (x, y, z=-66, -30, 36, t=4.876, P<0.05), left superior frontal gyrus(l-SFG) (x, y, z=-6, -33, 51, t=4.402, P<0.05), left inferior parietal lobe(l-IPL)( t=3.300, P<0.05) and right inferior parietal lobe(r-IPL) ( t=3.557, P<0.05) in fMDD group. Compared with the HC, FC decreased between the habenula and l-MOG (x, y, z=-48, -84, 3, t=-4.321, P<0.05) and left thalamus (x, y, z=-9, 3, 3, t=-3.971, P<0.05) in rMDD group, while FC increased between the habenula and left middle temporal gyrus(l-MTG)( x, y, z=-48, -39, 9, t=4.062, P<0.05), left supramarginal gyrus (x, y, z=-51, -15, 45, t=2.906, P<0.05), l-SFG (x, y, z=-24, -21, 39, t=3.044, P<0.05), l-IPL ( t=2.880, P<0.05) and r-IPL ( t=3.512, P<0.05) in rMDD group. (2) FC decreased in fMDD group between the habenula and right orbitofrontal cortex(r-OFC) ( t=-3.899, P<0.05) and l-MTG ( t=-4.023, P<0.05) than rMDD group, while FC increased between the habenula and left supramarginal gyrus ( t=4.157, P<0.05), l-SFG( t=3.327, P<0.05), left thalamus ( t=3.316, P<0.05) and l-IPL ( t=3.909, P<0.05) than rMDD group. (3)There was no significantly correlation between the HAMD 17 and the FC value changes among the different regions, and was marginal significantly correlation between duration of disease and the FC values from the habenula and l-MOG( r=0.328, P=0.063). Conclusion:Both fMDD and rMDD show abnormal FC between the habenula and default mode network, visual network and reward network, which may be related to the pathogenesis of depression. The FC of the habenula in rMDD still had not recovered to normal.