[Transformation of recombinant plasmid pRK 41 and amplification of full-length coding region of human brain myelin...

Transformation of the recombinant plasmid pRK41 containing 2.15 kd human brain myelin basic protein MBP)-coding sequence and 3' untranslated region (1.2 kb) into the E. coli JM109 was made by using Hanahan's method. Positive colonies were screened with digoxigenin oligo labelled rat brain MBP cDNA fragment (1.2 kb). To remove 3' untranslated region and obtain the full-length coding sequence of MBP cDNA, a pair of specific DNA primers was designed and synthesized. A 600 bp fragment was amplified from the recombinant plasmid, extracted from the positive colony by using polymerase chain reaction (PCR). The PCR fragment was isolated, and then digested with BamH I, Kpn I and BamH I + Kpn I. The results of restriction analysis indicate that the PCR amplified fragment is desirable and can be used directly to constract expression vectors.