Cloning and Expression of the Bacillus thruingiensis var. kurstaki HD-1 Crystal Protein gene in Eschelichia coli

The 44Md plasmid of Bacillus thruingiensis var. kurstaki HD-1(B. t k HD-1) was partially digested with Sau3AI and the fragments were cloned into E. coli HB101 on vector pBR322. Of 2, 950 clones with a recombinant pBR322, only one clone KC1 was determined to have the gene for crystal toxic proteins from the 44Md plasmid of B. t k HD-1 at the BamHI site of pBR322. The recombinant pBR322 was named pKC1 and its molecular size was 12kb. The KC1 produced a protein which was toxic to the silkworm and antigenically similar to the crystal toxic protein of B. t k HD-1. Also, electrophoretic mobility of the KC1 protein was apparently the same as that of the crystal toxic protein of B. t k HD-1.

Bacillus thruingiensis var. kurstaki HD-1의 내독소 단백질 유전자의 발현기작을 규명하기 위하여 이 균으로부터 내독소 단백질 유전자가 존재하는 것으로 확인된 29Md와 44Md plasmid를 분리한 후, Sau3AI 제어효소로 부분절단하고, pBR322 BamHI site에 ligation하여, E. coli HB101 strain에 transformation시켜, 3,000여개의 Ampr/Ters한 colony를 얻어 면역학적 방법과 살충적 검정으로 통해 재조합 균주 KC1을 얻었다. PKC1 plasmid DNA는 vector DNA를 포함하여 약 12kb 정도의 크기를 가지며, B. t k HD-1 내독소 단백질과 이동도가 같거나 일치하는 132kd, 117kd의 KC1 specific band 2개를 얻었다. KC1 cell extract를 첨식한 결과 약 80% 치사율을 보였다.