[Expression and purification of three fusion proteins containing a single B-cell epitope (beta5, beta9 or beta8) of human chorionic gonadotropin beta subunit].
The possibility of using a subunit or fragment of human chorionic gonadotropin (hCG) as an immunogen for birth control has been actively explored for many years. This protein homone is produced by the fertilized egg and is required for implantation of the blastocyst into the maternal uterus and the maitenance of pregnancy. In previous studies, several bio-synthesized hCG chimeric peptides (CP) that contain three linear B-cell epitopes (beta5, beta9 and beta8) of beta-hCG subunit together with various foreign 'promiscuous' T-cell epitopes were constructed and expressed as potential new hCG vaccine immunogens. In order to detect antibodies to each of the individual B-cell epitopes present in the animal antiserum raised against the hCG CPs, we decided to construct three recombinant proteins, each contains a single target B-cell epitope (betaE) of beta-hCG. Two sets of DNA fragments were chemically synthesized encoding the beta5, beta9 and beta8 epitopes (betaE) 45 approximately 52, 113 approximately 116 or 133 approximately 144 of beta-hCG subunit and were inserted into the downstream of streptavidin (Stv) gene in pTSA18 separately, with or without an extra TAA codon at the 3'-terminals of the genes. SDS-PAGE analysis revealed that only Stv-betaE (-beta5, -beta9 or -beta8) fusion genes set with the TAA codon can be expressed in E. coli BL21 (DE3) pLysS strain at high level after 1mM IPTG induction for 4 hours. Additionally, these fusion proteins can all be recognized by specific polyclonal antiserum (RS-4157) generated upon immunization with the loop peptide 38 approximately 57 of beta-hCG, monoclonal antibody (mAb) FB12 to beta9 epitope and mAb OT3A that specially recognizes reporter sequence 133 approximately 139 of beta8 epitope 137 approximately 144. Each of the proteins can be purified to 95% relative homogeneity using an improved method of preparative gel polyacrylamide gel electrophoresis. The yields were 5 mg per 1 L culture. The three target Stv-betaE fusion proteins will be useful in determining the immunogenicity of designed hCG CPs and hCG vaccines, including hCG DNA vaccines.
Xu
Wan-Xiang
WX
Department of Reproductive Biology, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China. xuwx@sippr.stc.sh.cn
He
Ya-Ping
YP
He
Yuan
Y
Wang
Li-Ting
LT
Sun
Zhi-Da
ZD
Ji
Zhao-Neng
ZN
Gu
Shao-Hua
SH
Chen
Jin-Zhong
JZ
Ying
Kang
K
Xie
Yi
Y
D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics / Q000276:immunology*; D018985:Epitopes, B-Lymphocyte / Q000235:genetics*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D019809:Streptavidin / Q000235:genetics; D014614:Vaccines, Synthetic / Q000276:immunology
D018997:Chorionic Gonadotropin, beta Subunit, Human; D018985:Epitopes, B-Lymphocyte; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D019809:Streptavidin