Bảng 3.3. Hoạt tính mSK-nonhis sau hoạt hóa.
Chất hoạt hóa Hoạt tính mSK-nonhis sau khi hoạt hóa ở 4°C, 6 h 0% glycerol (U/mg) % 10% glycerol (U/mg) % Triton X-100 (0,5%) 11208 ± 201 0,9 74279 ± 431 6,3 Tween 20 (1%) 73273 ± 43 6,2 95463 ± 2184 8,0 Tween 80 (1,5%) 35860 ± 1164 3,0 66081 ± 560 5,6 Khơng chất hoạt hóa 11877 ± 309 1 11877 ± 309 1
Kết quả hoạt hóa mSK-nonhis cũng tương tự với kết quả của Vũ Hồng Diệp (2010), Triton X-100 và Tween 20 bổ sung glycerol có khả năng hoạt hóa SK tốt nhất. Theo Vũ Hồng Diệp, khi có mặt glycerol, Triton X-100 hoạt hóa SK cao nhất, tăng 40 lần; khi khơng có glycerol tăng 12 lần. Khi có mặt glycerol, Tween 20 đã hoạt hóa SK gấp 35 lần và 10 lần so với hoạt hóa chỉ bằng đệm phosphate và khơng có glycerol [4].
KẾT LUẬN VÀ KIẾN NGHỊ Kết luận
1. Thiết kế thành cơng gene mã hóa streptokinase từ DNA tổng số của chủng
Streptococcus pyogenes DT7 trong vector biểu hiện pET21a(+) không đuôi histidine trong tế bào E. coli JM109(DE3)
2. Biểu hiện mSK-nonhis với năng suất đạt 72% protein tổng số, hoạt tính đạt 1275 U/ml tương đương 10200 U/mg tế bào tươi.
3. mSK-nonhis tinh sạch bằng siêu âm có hoạt tính cao nhất 11308 U/mg protein, được hoạt hóa bằng 1% Tween 20 bổ sung 10% glycerol có hoạt tính tăng 8 lần đạt 95463 U/mg protein.
Kiến nghị
1. Tiến hành loại bỏ nội độc tố vi khuẩn (lipopolysaccharide) ở dịch enzyme tinh sạch.
2. Thử nghiệm hoạt tính tan huyết của mSK-nonhis trên động vật thí nghiệm và tiến tới thử nghiệm tiền lâm sàng.
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PHỤ LỤC
Bảng 1. Hàm lượng protein các mẫu tinh sạch.
Mẫu Độ pha loãng (lần) OD (595 nm) HLPr tổng số mg/ml OD1 OD2 Chưa ts 5 0,516 0,52 1,070 ± 0,006 Dịch ts lần 1 (cặn 1) 1 0,486 0,471 0,198 ± 0,004 Dịch ts lần 2 (cặn 2) 1 0,453 0,451 0,188 ± 0,001 Dịch ts lần 3 (cặn 3) 1 0,422 0,422 0,176 ± 0 Dịch ts (cặn 4) 1 0,431 0,426 0,178 ± 0,001
HLPr: Hàm lượng protein; ts: tinh sạch.