病毒学报2016,20(4),393-398 DOI:10.165408/j.cnki.11-5866/n.2016.03.001

Generation of Human ScFv Antibodies for Antigenic SiteⅢof Rabies VirusGlycoprotein from Antibody-phage Libraries by Chain Shuffling

SUN Lina1 LIU Yang1 LI Chuan1 LI Dexin2 LIANG Mifang1
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School of Earth Sciences and Resources, China University of Geosciences, Beijing 100083, China

MEI Mingxiang, born in 1965, received his Ph.D. degree from China University of Geosciences in 1993. Now he is a professor at School of Earth Sciences and Resource, China University of Geosciences (Beijing), and is engaged in sedimentology and stratigraphy.

Accepted:2016-10-31

Funds: the Natural Sciences Foundation of China(grants No.41472090,40472065 and 49802012)

Abstract:To obtain neutralizing high affinity human recombinant antibodies for antigenic siteⅢ of rabies virus(RV)glycoprotein,we chose scFv phage display technology to optimize CR4098with chain shuffling. Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage libraries were constructed to replace CR4098light or heavy chain genes respectively by antibody genes derived from the blood of RV-vaccinated donors.After package by hyperphage,the chain shuffling scFv phage library was panned and selected by ELISA with purified rabies virus aG strain.The specific antibody was converted to full human IgG antibody with the VH/VK Express cassettes.Affinity and neutralizing test were performed to verify the function of the IgG molecules.Fourteen unique human ScFv antibodies specific for the glycoprotein of rabies virus were obtained by ELISA,IFA and DNA sequencing.Further tested showed that RV3A5has a high affinity of 2.8×10-9 M and high neutralizing activity to rabies virus both aG strains and CVS strains.Competitive ELISA showed that RV3A5and CR4098for antigenic Site III competed with each other,indicating that they had overlapping or shared the epitope.Our results provide more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.

Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage[ 1[1]俞永新.狂犬病和狂犬病疫苗[M].北京:中国医药科技出版社,2001.],Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage[ 2]。Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage[ 3]。由于RIG1注释:阿达啊飒飒大 Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage[ 4,5],Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage[ 6]。Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage[ 7]。

Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage[ Supplementary Material]。

title 2

1 VH/VK Express cassettes

To obtain neutralizing high affinity human recombinant antibodies for antigenic siteⅢ of rabies virus(RV)glycoprotein,we chose scFv phage display technology to optimize CR4098with chain shuffling. Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage libraries were constructed to replace CR4098light or heavy chain genes respectively by antibody genes derived from the blood of RV-vaccinated donors.After package by hyperphage,the chain shuffling scFv phage library was panned and selected by ELISA with purified rabies virus aG strain.The specific antibody was converted to full human IgG antibody with the VH/VK Express cassettes.

2 VH/VK Express cassettes

To obtain neutralizing high affinity human recombinant antibodies for antigenic siteⅢ of rabies virus(RV)glycoprotein,we chose scFv phage display technology to optimize CR4098with chain shuffling. Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage libraries were constructed to replace CR4098light or heavy chain genes respectively by antibody genes derived from the blood of RV-vaccinated donors.After package by hyperphage,the chain shuffling scFv phage library was panned and selected by ELISA with purified rabies virus aG strain.The specific antibody was converted to full human IgG antibody with the VH/VK Express cassettes.

3 VH/VK Express cassettes

To obtain neutralizing high affinity human recombinant antibodies for antigenic siteⅢ of rabies virus(RV)glycoprotein,we chose scFv phage display technology to optimize CR4098with chain shuffling. Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage libraries were constructed to replace CR4098light or heavy chain genes respectively by antibody genes derived from the blood of RV-vaccinated donors.After package by hyperphage,the chain shuffling scFv phage library was panned and selected by ELISA with purified rabies virus aG strain.The specific antibody was converted to full human IgG antibody with the VH/VK Express cassettes.[ Supplementary Audio]。

title 2

1、VH/VK Express cassettes

To obtain neutralizing high affinity human recombinant antibodies for antigenic siteⅢ of rabies virus(RV)glycoprotein,we chose scFv phage display technology to optimize CR4098with chain shuffling. Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage libraries were constructed to replace CR4098light or heavy chain genes respectively by antibody genes derived from the blood of RV-vaccinated donors.After package by hyperphage,the chain shuffling scFv phage library was panned and selected by ELISA with purified rabies virus aG strain.The specific antibody was converted to full human IgG antibody with the VH/VK Express cassettes.

2、VH/VK Express cassettes

To obtain neutralizing high affinity human recombinant antibodies for antigenic siteⅢ of rabies virus(RV)glycoprotein,we chose scFv phage display technology to optimize CR4098with chain shuffling. Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage libraries were constructed to replace CR4098light or heavy chain genes respectively by antibody genes derived from the blood of RV-vaccinated donors.After package by hyperphage,the chain shuffling scFv phage library was panned and selected by ELISA with purified rabies virus aG strain.The specific antibody was converted to full human IgG antibody with the VH/VK Express cassettes.[ Supplementary Video]。

3、VH/VK Express cassettes

To obtain neutralizing high affinity human recombinant antibodies for antigenic siteⅢ of rabies virus(RV)glycoprotein,we chose scFv phage display technology to optimize CR4098with chain shuffling. Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage libraries were constructed to replace CR4098light or heavy chain genes respectively by antibody genes derived from the blood of RV-vaccinated donors.After package by hyperphage,the chain shuffling scFv phage library was panned and selected by ELISA with purified rabies virus aG strain.The specific antibody was converted to full human IgG antibody with the VH/VK Express cassettes.。

To obtain neutralizing high affinity human recombinant antibodies for antigenic siteⅢ of rabies virus(RV)glycoprotein,we chose scFv phage display technology to optimize CR4098with chain shuffling. Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage libraries were constructed to replace CR4098light or heavy chain genes respectively by antibody genes derived from the blood of RV-vaccinated donors.After package by hyperphage,the chain shuffling scFv phage library was panned and selected by ELISA with purified rabies virus aG strain.The specific antibody was converted to full human IgG antibody with the VH/VK Express cassettes.( Table 1)。

To obtain neutralizing high affinity human recombinant antibodies for antigenic siteⅢ of rabies virus(RV)glycoprotein,we chose scFv phage display technology to optimize CR4098with chain shuffling. Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage libraries were constructed to replace CR4098light or heavy chain genes respectively by antibody genes derived from the blood of RV-vaccinated donors.After package by hyperphage,the chain shuffling scFv phage library was panned and selected by ELISA with purified rabies virus aG strain.The specific antibody was converted to full human IgG antibody with the VH/VK Express cassettes.[ Supplementary Figure1]。

Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage[ Supplementary Table 1]。Using pHAL14-CR4098as vector,the combinatorial shuffling scFv antibody phage( Figure1)。