Multiple (His-Pro) (mHP peptide)의 항당뇨 효과를 증명하기 위하여, 먼저 mHP peptide를 alkaline protease promoter (0.45 kb)와 신호서열 (87 bp)을 포함하는 pRBAS 분비벡터를 이용하여 고초균에서 분비 및 최적화 실험을 하였고, Insulin종인 RINm5F 세포에서 mHP peptide의 항당뇨 효과를 직접적으로 조사하였다. mHP peptide 유전자는 항당뇨 효과가 알려진 cyclo(His-Pro)를 만들기 위하여 His-Pro 서열이 반복된 oligonucleotide의 annealing에 의해 얻었고, 최종적으로 18 dipeptides (108 bp and 4.0 kDa)로 구축하였으며 multiple (His-Pro) (mHP peptide)로 명명하였다. mHP peptide 유전자는 개시코돈과 종결코돈, His tag 서열이 포함된 primer들로 PCR 증폭 후 pRBAS 분비벡터로 클로닝하였고 pRBAS-mHP (6.56 kb)로 명명하였다. mHP peptide의 분비 최적화를 위하여 고초균 LKS87에서 다양한 배양조건들로 조사하였고 그 결과, 분비된 mHP peptide는 3 L batch culture에서 가장 높게 측정되었으며 (약 59.6 μg/mL), 그 때의 조건은 30°C, PY medium, 그리고 carbon sources (특히, barley and glycerol)이었다. STZ (2 mM) (T1DM)를 처리한 RINm5F 세포에 mHP peptide (0.1 mg/mL)를 처리하자 세포활성 (10%)이 복구되었고, nitric oxide (NO) (35%)와 DNA fragmentation 현상 (90%)이 감소되었으며 Insulin (17%)이 더 높게 분비되었다. mHP peptide (4-12 mg/kg) 처리한 (C57BL/6J ob/ob) (T2DM)에선 대조군에 비해 Insulin（12％)이 증가했고, 혈당 (19%)이 감소했다. 이러한 결과들은 mHP peptide가 항당뇨 효과를 가지는 후보물질이 될 수 있음을 시사하고 있다.


이 때 얻어진 결과들은 다음과 같았다.
1. primer set를 사용하여 증폭한 multiple (His-Pro) peptide (mHP peptide)의 PCR 산물들을 pGEM-T easy vector에 클로닝하였다. 추정되는 클론들은 크기와 제한효소 처리를 통해 선별하였고, 분비벡터를 구축하기 위하여 사용하였으며 얻어진 클론을 pT-mHP라고 명명하였다. pT-mHP에서 잘라낸 mHP의 DNA 조각과 pRBAS vector를 ligation하여 E. coli XL1-blue MRF로 형질전환하였고, 얻어진 클론을 pRBAS-mHP라고 명명하였다. mHP 유전자가 포함된 pRBAS-mHP 재조합 플라스미드는 Bacillus subtilis에서 mHP를 발현 및 생산하기 위하여 구축되었다.

2. pRBAS-mHP 재조합 플라스미드가 형질전환 되어있는 E. coli XL1-blue MRF를 Spizizen‘s minimal medium method에 따라 Bacillus subtilis LKS87로 형질전환하였고, pRBAS-mHP가 형질전환 되어있는 Bacillus subtilis LKS87의 분비벡터를 Bacillus subtilis DNA와 PCR을 통하여 확인하였다. 결과적으로 agarose gel electrophoresis를 통하여 His-tag이 포함된 mHP (126 bp)를 확인하였다.

3. 분비벡터인 pRBAS-mHP를 Bacillus subtilis LKS87과 168로 도입하였다. 배양 조건에 따라 배양한 후, 상층액으로 만든 단백질 샘플들은 SDS-PAGE와 western blot에 의해 분석되었다. pRBAS-mHP 분비벡터가 포함된 모든 균주들은 mHP peptide를 생산하였지만, 오직 pRBAS만을 포함한 균주에서는 mHP peptide가 확인되지 않았다. mHP 분비는 Bacillus subtilis LKS87에서 가장 높은 발현 수준을 보였고, 또한 다른 LB배지나 MYP배지보다 PY배지에서, 37℃ 보다는 30℃에서 더 높은 발현 수준을 보였다. 결과적으로 pRBAS-mHP 분비벡터가 Bacillus subtilis LKS87, PY배지, 30℃에서 가장 mHP peptide가 높게 분비되었음을 알 수 있다.

4. High constitutive expression을 이용한 pRBAS 벡터의 시스템은 용해 상태로 높은 수준의 재조합 mHP peptide를 발현시키는데 사용되었고, SDS-PAGE와 western blot 분석은 Bacillus subtilis LKS87 상층액으로 분비된 재조합 mHP peptide를 보여주었다. 또한, 세포가 용해될 때 용해된 mHP peptide 부분은 상층액에 존재하며 세포 pellet에서는 보이지 않았다.

5. 배양액 1 mL 당 용해되어있는 mHP peptide의 양 (μg)은 SDS-PAGE 상의 mHP peptide 밴드의 상대적 함량에 대한 Bradford method를 사용함으로써 측정하였다. 결과적으로, 30℃, PY배지, 1% soy peptone, 3% barley 그리고 4% edible glycerol 배지 조건에서 pRBAS-mHP가 형질전환된 Bacillus subtilis LKS87에서 용해된 mHP peptide가 가장 높은 수준으로 분비되었다 (59.6 μg/mL).

6. mHP peptide는 24 h에서는 약간 분비되다가 48 h 배양을 하자 완전히 분비되기 시작했고 가장 구간 별 분비 증가량이 높았다. 그리고 지속적으로 mHP peptide 분비는 48-72 h에서 증가하였으며 72 h에서 최고로 높은 발현 수준을 보여주었다.

7. Streptozotocin (STZ)으로 인해 손상된 베타 세포의 기능장애와 아폽토시스에 대한 mHP peptide의 효과들은 Rat Insulinoma cell인 Insulin을 분비하는 RINm5F 세포에서 조사되었다. RINm5F 세포를 2 mM STZ로 처리하자 cell viability와 Insulin secretion은 대조군과 비교하였을 때 각각 약 49%와 58%로 감소하였지만, mHP peptide를 처리하자 cell viability와 Insulin secretion에 대한 RINm5F 세포의 활성이 처리하지 않은 대조군보다 약 59%와 68%로 복구되었다 (각각 10% 증가). 그리고 mHP peptide는 STZ로 인한 nitric oxide (NO) 발생 (35%)을 감소시킴으로써 독성으로부터 세포를 상당히 보호하였다. 더구나, mHP peptide 처리는 RINm5F 세포에서의 STZ로 인한 아폽토시스적 DNA fragmentation 현상 (90%)을 완화시켰고, STZ만 처리한 대조군보다 Insulin이 더 높게 분비되었다 (17%). 이것은 mHP가 STZ의 산화적 스트레스로 인해 발생된 아폽토시스적 세포 죽음으로부터 세포를 보호할 수 있다는 가능성을 보여준다.

8. T2DM 동물 연구를 하기 위해서 ob/ob mice (C57BL/6J ob/ob)에 mHP peptide를 6주 동안 체중에 따라 4-12 mg/kg 수준으로 2주씩 처리하였다. 동물들은 12:12 시간 동안 낮과 밤이 순환되며 온도 (55 ± 5 % 습도 23 ± 2℃)가 조절되는 스테인레스 철 케이지에 개별적으로 수용하였고 상업적인 펠릿 먹이 (Sam Yang Co., Seoul, Korea)와 물을 실험기간 동안 제공하였다. 모든 동물들은 무작위로 대조군과 실험군 2 그룹으로 나누었고 혈당수치가 300 mg/dL(16.7 mmol/L) 이상의 쥐들을 당뇨병으로 간주했다. 체중증가와 음십섭취는 주기적으로 측정했으나 대조군과 실험군의 차이가 크지 않았고, plasma에서의 Insulin level은 12% 정도 증가하였다. 또한, 1-4시간 동안 금식 후 혈당측정기 (Lifescan, Milpitas, CA, USA)를 사용하여 혈당수치를 측정하였고, 이 때 쓰인 혈액은 쥐 꼬리의 하부를 바늘로 찔러 얻었다. 그 결과 대조군에 비해 실험군의 혈당이 평균적으로 12%, 최대 19% 정도 감소하였다. 이 실험들은 대구대학교의 동물 보호와 이용 연구실 위원회에 의하여 승인받았다. 모든 절차들은 건강국제기관에서 출판된 ‘동물 실험실에서의 보호와 이용 가이드’에 따라서 수행했다.

9. 마지막으로, 이러한 결과들은 mHP peptide가 STZ로 인한 세포 독성과 아폽토시스에 대한 보호 및 치료의 후보물질로서 이용될 수 있음을 보여주고 있다.

To verify anti-diabetic effect of multiple (His-Pro) peptide (mHP peptide), the mHP peptide was first secreted and optimized using the secretion vector, pRBAS with alkaline protease gene promoter (0.45 kb) and the signal sequence (87 bp) in Bacillus subtilis and directly the anti-diabetic effect of the mHP peptide was investigated in Insulinoma cell, RINm5F cell line. The mHP gene was obtained by annealing oligonucleotides with repeated His-Pro sequence and finally was constructed as 18 dipeptides (108 bp and 4.0 kDa) coding gene, named multiple (His-Pro) peptide (mHP peptide) to make cyclo(His-Pro) known to be anti-diabetic effects. The region encoding the mHP gene was subcloned into the pRBAS secretion vector (E. coli-Bacillus shuttle vector) after PCR amplification using the designed primers including initiation and termination codons and His tag, named pRBAS-mHP (6.56 kb). To optimize secretion of the mHP peptide, various culture conditions were investigated in Bacillus subtilis LKS87. As a result, the secreted mHP peptide was maximally measured (approximately 59.6 μg/mL) in 3 L batch culture and the highest secretion was achieved at 30°C, PY medium, and carbon sources (particularly barley and glycerol). In the RINm5F cells treated with 2 mM STZ (T1DM), the mHP peptide treatment (0.1 mg/mL) restored the cell viability (10%) and reduced the nitric oxide (NO) generation (35%) and DNA fragmentation (90%). And also, Insulin secretion level was increased to 17% higher than in STZ-treated RINm5F cells. The ob/ob mice (C57BL/6J ob/ob) (T2DM) were treated with mHP at a level of 4-12 mg/kg of body weight for 6 weeks. Plasma Insulin level (12%) was increased and blood glucose level (19%) was reduced than control group. These results suggest that the mHP peptide with His-Pro repeats could be a candidate material for anti-diabetic agent against STZ-induced diabetes.

The obtained results were as followings:
1.The PCR products, amplified multiple (His-Pro) peptide (mHP peptide) using primer set were cloned into pGEM-T easy vector. The putative clones were screened by size selection and restriction enzyme digestion and used for the construction of secretion vectors. The obtained clone was named pT-mHP. The resulting fragments of pT-mHP were ligated to pRBAS vector and transferred into E. coli XL1-blue MRF. The obtained clone was named pRBAS-mHP. The pRBAS-mHP recombinant plasmids which contain mHP gene have been constructed to express and secrete the mHP peptide in Bacillus subtilis.

2.The obtained in E. coli XL1-blue MRF harboring pRBAS-mHP recombinant plasmids were transferred into Bacillus subtilis LKS87 by Spizizen''s minimal medium method. Secretion vector of Bacillus subtilis LKS87 harboring pRBAS-mHP was confirmed by preparation of Bacillus subtilis plasmid DNA and PCR. As a result, confirmed mHP peptide contained His-tag (126 bp) by agarose gel electrophoresis.

3.The secretion vector, pRBAS-mHP was introduced into Bacillus subtilis LKS87. After incubation in culture condition, supernatant was analyzed and protein samples were subjected to SDS-PAGE and Western blot analysis. All strains harboring the secretion vector was produced mHP peptide, while the protein doesn''t be detected in strain with only vector, pRBAS. mHP peptide secretion was showed the highest expression level in Bacillus subtilis LKS87. And also, the expression levels were greater in the PY medium better than other medium (LB, MYP) and showed higher expression level at 30℃ than at 37℃. Consequently, secretion vector pRBAS-mHP was exhibited the highest mHP peptide in Bacillus subtilis LKS87, in PY medium at 30℃.

4.Using High Constitutive Expression, system of pRBAS vector were facilitated high-level expression of the recombinant mHP peptide in a soluble form. SDS?PAGE and Western blot analysis showing the secretion of the recombinant mHP peptide in supernatant of Bacillus subtilis LKS87. When cells were lysed, the soluble mHP peptide was recovered in the soluble fraction (supernatant), and not visible in the cell pellet.

5.The amount (μg) of soluble mHP peptide per 1 mL culture was estimated from the Bradford method by correcting it with relative percentage of mHP peptide band on SDS-PAGE. As a result, the highest level of secretion was secreted (59.6 μg/mL) soluble mHP peptide in B. subtilis LKS87 harboring pRBAS-mHP at 30℃, in PY medium, 1% soy peptone, 3% barley and 4% edible glycerol medium composition.

6. The secretion of mHP peptide was slightly secreted at 24 h and completely appeared 48 h after incubation. The secretion quantity was many increased start 48 h. And, continuously increased at 48 h and 72 h. The secretion of mHP peptide was showed the highest expression level at 72 h.

7. The effects of mHP peptide on streptozotocin (STZ)-induced β-cell dysfunction and apoptosis were investigated in rat Insulinoma cells (RINm5F) secreting Insulin. When the RINm5F cells were treated with 2 mM STZ, cell viability and Insulin secretion decreased to approximately 49% and 58% compared to that of the control cell, relatively. However, mHP peptide treatment restored cell viability and Insulin secreting activity of RINm5F cells to approximately 59% and 68% of the untreated control cells (10% increase, relatively). And also, mHP peptide significantly protected the cells from STZ-mediated cytotoxicity by reductions of nitric oxide (NO) production (35%) induced by STZ. Moreover, mHP peptide treatment also attenuated STZ-induced apoptotic DNA fragmentation (90%) in RINm5F cells, and increased Insulin secretion (17%), indicating that mHP peptide could prevent the cells from apoptotic cell death induced by oxidative stress of STZ.

8. For T2DM animal experiments, the ob/ob mice (C57BL/6J ob/ob) were treated with mHP at a level of 4-12 mg/kg of body weight for 6 weeks. Animals were housed individually in steel cages at a controlled temperature (23±2°C with 55±5% humidity) under a light:dark cycle (12:12h). A commercial pellet diet (Sam Yang Co., Seoul, Korea) and water were provided during the experiment period. All animals were randomly divided into each group consisting of control and mHP-animals, and the ob/ob mice with a blood glucose level of 300 mg/dL (16.7 mmol/L) were considered to be diabetic. Body weight gain and food intake were periodically measured. After fasting for 1-4 h, blood glucose levels were measured using one drop of blood by piercing the underside of the tails of ob/ob mice with a glucometer (Lifescan, Milpitas, CA, USA). The ob/ob mice (C57BL/6J ob/ob) (T2DM) were treated with mHP peptide at a level of 4, 8 and 12 mg/kg of body weight for 2 weeks, respectively. Plasma Insulin level (12%) was increased and blood glucose level (19%) was reduced than control group. These experiments were approved by the Committee for Laboratory Animal Care and Use of Daegu University. All procedures were conducted according to the “Guide for the care and use of laboratory animals” published by the National Institutes of Health.


9. Finally, these results suggest that the mHP peptide could be used as a candidate material for the protective and therapeutic agent against STZ-mediated cytotoxicity and apoptosis.