A human BAC clone containing the whole human CD200 gene was used as a template for long-distance PCR to obtain a region bearing exon 1, intron 1, exon 2, intron 2 and exon 3 of the human CD200. Two mixtures were prepared: mix 1 (20 ml) contained 0.1 mg of DNA template, 0.5 mM dNTP mix and 0.5 mm of sense and antisense primers; mix 2 (30 ml) included elongase enzyme mix and 1Â long-distance PCR buffer A and B provided by the manufacturer (the ratio of buffer A and B is 1:4). The sense primer started with the NotI cleavage site and the antisense primer with the SalI site. The sequences of the primers were shown in Table 1 . Mix 1 and mix 2 were combined on ice and subject to PCR under the following condition: 94 C for 1 min followed by three cycles at 94 C for 30 s, 59 C for 30 s, 69 C for 20 min, and then 29 cycles of 94 C for 30 s, 69 C for 20 min. The final extension was 69 C for 15 min. The 12-kb CD200 fragment was displayed on 0.7% TAE-agarose gel and purified using QIAEX II Agarose Gel Extraction Kit following the manufacturer's instruction. For more efficient elution of the large size DNA, the final incubation time was extended to 30 min at 60 C. The gel-purified DNA fragment was verified by restriction enzyme digestion with BamHI, BglII, EcoRI and HindIII, respectively, and DNA sequencing. For ligation to pcDNA 3.0 expression vector, the CD200 fragment was digested with NotI and SalI. Meanwhile, pcDNA 3.0 expression vector was digested with NotI and XhoI. Afterwards, pcDNA 3.0 vector was further dephosphorylated to remove the 5 0 phosphoryl group, preventing the vector from selfligation. The enzyme-treated CD200 fragment and pcDNA 3.0 were ligated, at a molar ratio of 3:1, using 