A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling ...A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α_A-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.展开更多
In order to improve the functional affinity of the humanized VH single domain antibody against human lung cancer, the genes coding the homogenous dimers dihu3D3Vn and tetramers tehu3D3VH were constructed by fusing the...In order to improve the functional affinity of the humanized VH single domain antibody against human lung cancer, the genes coding the homogenous dimers dihu3D3Vn and tetramers tehu3D3VH were constructed by fusing the SV5-Cys short peptide and p53 tetramefization structural domain gene to hu3D3VH gene via recombinant PCR technique, respectively. Then, the dihu3D3VH and tehu3D3VH genes were cloned to the prokaryotic expression vector pET-22b（ ＋ ） and expressed in E. coli BL21 （DE3）. The proteins expressed were purified through Ni^2＋ -affinity chromatographic column. Meanwhile, the hu3D3VH, dihu3D3VH and tehu3D3VH proteins were labeled with FTTC, and their reactivity with antigen and specificity were analyzed by immunofluorescence assay. As to their functional affinities, it was analyzed and compared by flow cytometry. The results indicated that these two genes were expressed as monomers and mainly as inclusion bodies. After purification and renaturation, there were about 50% of dimers and 70% of tetramer remaining in the protein solution. In addition, the dihu3D3VH and tehu3D3VH proteins still remained the reactivity with antigen and specificity of hu3D3VH protein, and their functional affinities were increased about 60% or 100% respectively, compared with those of hu3D3VH protein. It is evident that the functional affinity of hu3D3VH protein can be greatly improved by increasing its binding valency.展开更多
Immune imprinting,or original antigenic sin,challenges the control of evolving severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).How the quantity and quality of pre-existing immunity modulate the recall anti...Immune imprinting,or original antigenic sin,challenges the control of evolving severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).How the quantity and quality of pre-existing immunity modulate the recall antibody response upon re-exposure remains poorly understood.We immunized hamsters with a recombinant ancestral spike protein(S-2P)formulated with one of four distinct adjuvants to assess the impact of adjuvant-induced immunity on protective efficacy.Furthermore,we analyzed the modulatory effect of the adjuvant on the potency,breadth,and evolution of antibody responses after homologous viral challenge.We found that vaccination with Montanide ISA720-adjuvanted S-2P(S-2P:ISA720)not only induced higher initial neutralizing antibody titers and conferred stronger protection but also established a pre-existing immune state capable of cross-neutralizing the antigenically distant Omicron BA.1 variant.In contrast,aluminum hydroxide adjuvanted S-2P(S-2P:Al)elicited comparatively lower neutralizing titers and showed no cross-neutralization against BA.1.Following viral challenge,the S-2P:ISA720 group exhibited significant affinity maturation toward conserved epitopes,which markedly enhanced cross-neutralization against BA.1 without increasing neutralizing titers or affinity against the homologous strain or the antigenically related Delta variant.Conversely,the S-2P:Al group mounted a narrow,imprinting-facilitated response,characterized by boosting of strain-specific antibodies without substantial improvement in BA.1 neutralization.These findings suggest that in S-2P:ISA720-immunized animals,high-affinity antibodies mediate epitope masking of immunodominant sites,thereby redirecting responses toward subdominant conserved epitopes with cross-neutralizing potential.In conclusion,our study demonstrates that adjuvants can critically guide recall immune responses toward breadth and affinity maturation,offering a rational strategy for developing next-generation vaccines against SARS-CoV-2 and other variable pathogens.展开更多
文摘A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α_A-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.
文摘In order to improve the functional affinity of the humanized VH single domain antibody against human lung cancer, the genes coding the homogenous dimers dihu3D3Vn and tetramers tehu3D3VH were constructed by fusing the SV5-Cys short peptide and p53 tetramefization structural domain gene to hu3D3VH gene via recombinant PCR technique, respectively. Then, the dihu3D3VH and tehu3D3VH genes were cloned to the prokaryotic expression vector pET-22b（ ＋ ） and expressed in E. coli BL21 （DE3）. The proteins expressed were purified through Ni^2＋ -affinity chromatographic column. Meanwhile, the hu3D3VH, dihu3D3VH and tehu3D3VH proteins were labeled with FTTC, and their reactivity with antigen and specificity were analyzed by immunofluorescence assay. As to their functional affinities, it was analyzed and compared by flow cytometry. The results indicated that these two genes were expressed as monomers and mainly as inclusion bodies. After purification and renaturation, there were about 50% of dimers and 70% of tetramer remaining in the protein solution. In addition, the dihu3D3VH and tehu3D3VH proteins still remained the reactivity with antigen and specificity of hu3D3VH protein, and their functional affinities were increased about 60% or 100% respectively, compared with those of hu3D3VH protein. It is evident that the functional affinity of hu3D3VH protein can be greatly improved by increasing its binding valency.
基金supported by the National Key Research&Development Program of China(2022YFC2304100)the Major Research Plan of the National Natural Science Foundation of China(92169206).
文摘Immune imprinting,or original antigenic sin,challenges the control of evolving severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).How the quantity and quality of pre-existing immunity modulate the recall antibody response upon re-exposure remains poorly understood.We immunized hamsters with a recombinant ancestral spike protein(S-2P)formulated with one of four distinct adjuvants to assess the impact of adjuvant-induced immunity on protective efficacy.Furthermore,we analyzed the modulatory effect of the adjuvant on the potency,breadth,and evolution of antibody responses after homologous viral challenge.We found that vaccination with Montanide ISA720-adjuvanted S-2P(S-2P:ISA720)not only induced higher initial neutralizing antibody titers and conferred stronger protection but also established a pre-existing immune state capable of cross-neutralizing the antigenically distant Omicron BA.1 variant.In contrast,aluminum hydroxide adjuvanted S-2P(S-2P:Al)elicited comparatively lower neutralizing titers and showed no cross-neutralization against BA.1.Following viral challenge,the S-2P:ISA720 group exhibited significant affinity maturation toward conserved epitopes,which markedly enhanced cross-neutralization against BA.1 without increasing neutralizing titers or affinity against the homologous strain or the antigenically related Delta variant.Conversely,the S-2P:Al group mounted a narrow,imprinting-facilitated response,characterized by boosting of strain-specific antibodies without substantial improvement in BA.1 neutralization.These findings suggest that in S-2P:ISA720-immunized animals,high-affinity antibodies mediate epitope masking of immunodominant sites,thereby redirecting responses toward subdominant conserved epitopes with cross-neutralizing potential.In conclusion,our study demonstrates that adjuvants can critically guide recall immune responses toward breadth and affinity maturation,offering a rational strategy for developing next-generation vaccines against SARS-CoV-2 and other variable pathogens.
