HVR1-10# and 11# were sequences for the mimotope R9 and M122 respectively (Puntoriero et al[14], 1998), and HVR1-12# reported by Watanabe. Open in a separate window Figure 2 Derivation of the Chinese consensus sequence. a 27 HCV positive panel. Recombinant HVR1s of No. 1, 2, 4, and 8# showing broad cross-reactivities and complementarity with each other, were selected for the ligation elements. The chimera containing these 4 HVR1s was highly expressed in and to protect chimpanzees from HCV infection in vivo[8-10]. The HVR1 sequence is highly variable, and is the greatest obstacle for the vaccine development and immune therapy[11,12]. However, the highly variable HVR1s have been shown to have some cross-reactivities with each other, indicating that a broadly cross-reactive HVR1 peptide or their cocktails are helpful to solving the problem[13]. Data were accumulated in this study all over the world[14-17]. In China, HVR1 sequences of different HCV isolates have been reported by many authors, but few studies were on HVR1 cross-reactivity. Integrating the HVR1 sequences reported in China together with some published mimotopes, 12 representative HVR1 sequences were selected using bioinformatics technology. All of the representative HVR1 ASP 2151 (Amenamevir) sequences were cloned and expressed, and their cross-reactivity was studied with a panel of 27 HCV positive sera. Finally we obtained an HVR1 fusion antigen broadly cross-reactive with the HCV-infected sera. MATERIALS AND METHODS Human sera Samples of HCV-infected sera were obtained from blood donor applicants in Beijing Red Cross Blood Center and from chronic HCV-infected patients from 302 Hospital of PLA. All were positive for HCV antibodies using the 2nd-generation ELISA kit. (Ortho Diagnostics, Raritan N.J). Selection of representative HVR1 sequences All of the HVR1 sequences published in China were loaded into database and their consensus sequence was obtained by BASIC program according to the frequency of amino acid residues. All of these HVR1 sequences were divided into several groups according to their alignment, and one sequence was chosen as the representative from each group. All of the work above was operated on Goldkey (a molecular biology software developed by our institute). Some HVR1 sequences or mimotopes published were chosen as representative ones for their high cross-reactivity with sera of HCV infected patients from other countries. Construction of expression plasmid HVR1-1 # to 12 The representative HVR1 sequences were modified considering the Escherischia coli’s favorable ASP 2151 (Amenamevir) codon usage. The coding genes were synthesized chemically and to facilitate further ligation, two linkers with a specially designed restriction endonuclease site were incorporated into their N- and C- terminals respectively. The N terminal arm is F1 (5′-gcctcgagggtggtggatct-3′), The C terminal arm is R1 (5′-gctctagaacctccaccact-3′). The fragments were digested with I and I, while the plasmid pBVIL1-HVR1-2#, was chosen as the donor of pattern, amplified using constant primer F2 (5′-gcactagtggtggtggatct-3′) and R2 (5′-cgggatccttaggaagacacaaa-3′) which annealed to C-terminal of IL1. The PCR product was digested with I and I, and inserted into the digested vector, pBVIL1-HVR1-1#. Owe to the same cohesive end of the endonuclease I and I, the digested PCR fragment could ASP 2151 (Amenamevir) accurately linked to the digested plasmid and the new ligated site could be digested by neither of them. Open in a separate window Figure 1 Four different HVR1 gene fragments were cloned on pBVIL-1. HVR1-2 gene fragment was ligated with pBVIL-1-HVR1-1 (pHVR1#). The new pHVR1+2 had the same site with pHVR1#, and the HVR1-4# gene fragments could be ligated by the same way. After 3 cycles, the Capn2 chimera HVR1 plamid was constructed. The pBVIL1-HVR1-1+2# had the same enzyme sites with pHVR1# and so it could be used as a new vector and connected with other HVR1 gene fragments. In this way, the pBVIL1-chimeric-HVR1 was constructed to contain four HVR1 genes, HVR1-1#, HVR1-4#, HVR1-6# and HVR1-8#. Purification of representative HVR1-1-12# and the chimeric antigen The plasmids carrying HVR1 fragments were transformed into HB101 as routine, and were examined for their orientation and nucleic acid sequences. The transformed HB101 was grown overnight, diluted 1:20 with fresh LB-medium and further incubated at 37 C to an OD600 of 0.6. After induction for 4 h at 42 C, the bacteria were harvested by centrifugation, and lysed by sonication. All of the recombinant proteins existed in inclusion bodies, and could be dissolved in a solution containing 8 M urea. The recombinant proteins were isolated and purified consecutively by Q-Sepharose-FF and Sephadex G50 chromatography. ELISA Microplates were coated with 0.3 g recombinant HVR1 peptide in 100 mM phosphate buffer (pH7.4) by incubation overnight at 4 C. The plates were then blocked with the phosphate buffer containing 0.2% BSA at 4 C for 3 h, and then incubated with 100 L of the serum sample 1:10 diluted with a sample buffer (100 mM sodium phosphate buffer, pH7.5 containing 10% goat serum and 0.05% Tween) at 37 C for 1 h. After being washed for five times with 100 mM phosphate buffer (pH7.5) containing 0.05% Tween, the plates were then incubated.