The creatine kinase plays an important role in bovine energy metabolism. When inflammation occurs, there will be a locally-specific increase of CK. According, the creatine kinase may serve as a potential marker of bovine diseases. In this study, a total of 54 mutually overlapped primers were designed and synthesized according to the published amino acid sequence of bovine creatime kinase isoenzyme CK-BB and the codon usage preferred by E. coli (BL21). A total of 6 DNA fragments of CK-BB gene, including CK-BB1, CK-BB2, CK-BB3, CK-BB4, CK-BB5 and CK-BB6, were synthesized by assembled PCR assay. Using the mixture of the 6 fragments as template, the second round PCR was conducted with head primer ck-1 and tail primer ck-54, and the amplifying product was connected with vector pET28b. The positive recombinants were subsequently identified, cloned and sequenced. A correct synthetic CK-BB gene was obtained by proofreading PCR assay, which lays a foundation for large-scale preparation of bovine isoenzyme CK-BB.%牛肌酸激酶是重要的能量代谢酶，尤其是在机体发生炎症时，会在局部存在特异性升高，具有潜在的疾病标志物作用。根据牛肌酸激酶同功酶CK-BB的氨基酸序列及大肠杆菌（BL21）密码子的偏爱性，设计合成54条互相重叠的引物，通过组装PCR分别合成CK-BB的6个片段CK-BB1、CK-BB2、CK-BB3、CK-BB4、CK-BB5和CK-BB6；再以基因头ck-1和基因尾ck-54为引物，以CK-BB1、CK-BB2、CK-BB3、CK-BB4、CK-BB5和CK-BB6混合物为模板进行第2轮扩增；将得到的产物连接到pET28b载体上，挑取重组子测序。通过PCR扩增对人工合成的基因进行校正，得到完全正确的CK-BB基因，为重组牛肌酸激酶同功酶CK-BB的规模化制备奠定了基础。
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