The purified genomic DNA from $\underline{Streptococcus equisimilis}$ was digested partially with PstⅠ and eluted by electrophoresis to ten fractions according to the size.
Fractions 3, 4, 5 and 6 corresponding 7.4kb, 6.7kb, 4.4kb and 3.5kb were ligated with a vehicle, pBR322 which was digested with PstⅠ and dephosphorylated with BAP. The ligated DNA was transformed into competent $\underline{E. coli}$ HB101 cells and selected transformants showing streptokinase activity by the method of the caseinolytic activity of activated plasminogen.
Around 15,000 colonies were appeared on the selection media having tetracycline indicating the colonies are transformants. These transformed colonies were tooth-picked and plates with developed colonies were overlayed with 9m1 of 50mM Tris.Hcl, PH8.1/150mM Nacl, containing 90mg agar, 100g of human plasminogen, and 2ml of Skim milk.
Among the 15,000 transformants tested 26 colonies showed clear halo indicating the transformants contain streptokinase gene.
The insert size of the plasmid carrying the streptokinase gene was a 2.5kb, 4.3kb and 5.8kb respectively and designated as pDC2.5, pDC3.4 and pDC5.8. The 2.5kb fragment of pDC2.5 was a part of pDC4.3, pDC5.8 hybrid plasmids.
The restriction maps of all 3 hybrid plasmids were constructed by digesting with PstI, PvuII, SalI, HindIII, AvaI, BamHI, EcoRI, ClaI, respectively.
It was found that $\underline{E. coli}$ HB101 colonies transformed with three hybrid plasmid produced caseinolysis when tested in any state of growth.
Quantitatively, the smaller the insert size, the much product streptokinase was produced.
By means of purifying the proteins, I performed the polyacryl-amide gel electrophoresis, ε-ACP (ε-amino caproic acid) inhibition test and soybean trypsin inhibitor inhibition test and it was thought that the cloned gene was streptokinase gene.
$\underline{S. equisimilis}$ 에서 분리한 chromosomal DNA를 제한 효소 Pst I 으로 자르고 역시 제한효소 Pst I 와 BAP 를 처리한 유전인자 전달체 pBR 322 와 붙인뒤 이것을 대장균에 형질 변환 시키고, casein 과 plasminogen 을 처리함 으로써 casein 의 분해 능력이 생긴 균을 찾아 내었다. Tetracycline 이 있는 선택 배지에 약 15,000 개의 형질 변환균 들이 나타났으며, 이 균들을 LB 배지에 옮겨 casein/plg overlay test를 수행한 결과 26 개의 균이 SK 활성도를 보임을 알수 있었다. 새로 조합된 유전인자 전달매체를 pDC2.5, pDC4.3, pDC5.8 로 명명하였으며, 대장균 내에서 안전하게 유지되고, 세포 외부로 효소를 생성했다. pDC2.5, pDC4.3, pDC5.8은 여러가지 제한효소로 자른 결과 가장 작은 삽입 부위를 갖고 있는 DNA 인 pDC 1 은 Sal I, Pvu II, Hind III 의 제한효소 자리를 갖고 있음이 밝혀졌다.
$\underline{S. equisimilis}$ 와 pDC2.5, pDC4.3, pDC5.8를 갖는 대장균 사이의 SK 활성도에 관해 비교해 본 결과, 그중 가장 높은 활성도를 보이는 pDC 1 의 경우 $\underline{S. equisimilis}$ 보다 약 1.8 배의 활성도를 나타내었다. 이 결과로써 SK 활성도를 높이기 위해서 새로운 SK 유전자를 가지는 전달체의 개발과 좋은 숙주의 선택에 관한 연구가 계속되어야 하겠다.
