摘要
猪乳铁蛋白肽(Porcine lactoferricin -20, LFP-20)是来源于猪乳铁蛋白N端含20个氨基酸残基的阳离子抗菌肽。本研究以LFP-20为模板,通过分子设计对其进行改良,在此基础上研究了改良肽的抗菌活性和安全性,比较了改良抗菌肽LF-2、LF-4、LF-6与LFP-20在膜作用机制方面的差异；通过建立小鼠腿肌和腹腔大肠杆菌染菌模型,研究了改良抗菌肽LF-2、LF-6对小鼠感染大肠杆菌的保护作用,同时探讨了改良抗菌肽对健康小鼠和染菌小鼠免疫功能的影响；进一步利用大肠杆菌和毕赤巴斯德酵母表达系统成功表达了改良抗菌肽LF-6。主要研究结果如下：
1.猪乳铁蛋白肽的分子改良及改良肽的筛选
在分析LFP-20理化性质、氨基酸组成并对其进行结构预测的基础上,采用去除分子内二硫键、改变疏水性和芳香族氨基酸比例等策略对猪乳铁蛋白肽LFP-20进行分子改良,获得了8种改良肽。对改良肽抗菌活性研究结果表明：与模板肽LFP-20相比,改良肽LF-2、LF-4和LF-6对革兰氏阴性菌大肠杆菌、铜绿假单胞菌、猪霍乱沙门氏菌和鼠伤寒沙门氏菌的抗菌活性提高了2-64倍,对革兰氏阳性菌金黄色葡萄球菌和表皮葡萄球菌的抗菌活性提高了2-8倍,其中,以改良肽LF-6的抗菌活性最好。与模板肽LFP-20相比,在0～32μg/mL范围内三种改良肽对人和猪的红细胞溶血率没有显著增加(p>0.05)。与模板肽LFP-20相比,0～50μg/mL的改良肽LF-2、LF-4、LF-6对人和猪的外周血单核细胞增殖没有显著差异(p>0.05)；在0-32μg/mL范围内,除8μg/mL和16μg/mL的LF-4以外,改良肽LF-2、LF-4、LF-6对外周血单核细胞的细胞毒性也没有显著增加(p>0.05)。
2.猪乳铁蛋白肽及改良肽的膜作用机制研究
扫描电镜和透射电镜观察LFP-20及其改良肽LF-2、LF-4、LF-6对E.coli和S.aureus细胞形态影响结果表明,1×MIC的LFP-20及三种改良肽作用细菌30 min后,对细菌细胞膜结构均有破坏作用,能够导致细胞膜产生不同程度的突起或破损,菌体细胞壁及细胞膜断裂、细胞内容物泄漏,细胞质电子密度明显降低,表明细菌细胞膜是这些抗菌肽作用的重要靶点。在相同浓度下,三种改良肽LF-2、LF-4、LF-6对E.coli和S.aureus细胞膜产生的去极化作用明显强于模板肽LFP-20,更容易导致细胞膜电势的破坏。8μg/mL～32μg/mL的三种改良肽对E.coli细胞外膜渗透性的增强作用均显著高于模板肽LFP-20(p<0.05)；与LFP-20、LF-2、LF-4相比,LF-6导致E. coli细胞内膜渗透性的增加作用最为迅速、明显。LFP-20、LF-4和LF-6对DPX与LPS结合的最大抑制率与相近,分别为68%、56%和51%,达到50%抑制率时所需的LF-4和LF-6浓度却明显低于LFP-20,分别为7.96μg/mL和6.91μg/mL,而LFP-20达到50%抑制率时所需的浓度为17.30μg/mL; LF-4、LF-6置换DPX-LPS分子中DPX,结合LPS的能力稍弱于LFP-20,但实现与LPS最大结合所需肽浓度却明显低于LFP-20。16μg/mL和32μg/mL的四种抗菌肽对脂质体膜具有相似的破坏潜能,但与模板肽LFP-20相比,64μg/mL的三种改良肽对脂质体膜PC:PG(1：1)和PG呈现了更强的破坏作用,引发了40%以上钙黄绿素的释放。上述研究结果揭示,膜破坏机制是LFP-20及其三种改良肽对E.coli和S.aureus的主要杀菌机制；与LFP-20相比,LF-2、LF-4、LF-6提高的抗菌活性可能它们增强的对细菌细胞膜去极化作用、对细菌细胞内外膜渗透性、更容易与LPS结合以及对脂质体膜的破坏潜能密切相关。
3.猪乳铁蛋白肽及改良肽对小鼠感染大肠杆菌保护作用的研究
在研究LFP-20及其改良肽LF-2、LF-6对ICR小鼠急性毒性的基础上,建立了ICR小鼠腿肌和腹腔大肠杆菌感染模型,比较了LFP-20与改良肽LF-2、LF-6抵抗小鼠感染大肠杆菌的能力。急性毒性研究结果表明,LFP-20、LF-2、LF-6对ICR小鼠的LD5o分别为34.25 mg/kg、6.54 mg/kg和口29.52 mg/kg。腿肌感染E.coli K88试验结果表明,2mg/kg和口8 mg/kg的三种抗菌肽对小鼠腿肌染菌都具有预防效果,除2 mg/kg LFP-20外,另外五种剂量的抗菌肽均显著降低了小鼠腿肌E.coli活菌数(p<0.05)；其中,8 mg/kgLF-6对小鼠腿肌染菌的抑制效果最为显著(p<0.05),该组小鼠腿肌匀浆组织中的活菌数为3.85士0.24(lg CFU/g)。预防腹腔感染E.coli K88试验结果表明：与E.coli组相比,2 mg/kg和8 mg/kg的三种抗菌肽对染菌小鼠腹腔液、肝脏和肠系膜淋巴结感染的大肠杆菌都具有显著的抑制效果(p<0.05),其中以8 mg/kg LF-6的抑菌效果最好,其腹腔液、肝脏和肠系膜淋巴结中大肠杆菌活菌数分别为1.18±0.10(lg CFU/mL)、3.85±0.24 (lg CFU/g)和3.00±0.15(lg CFU/g)。E.coli组盲肠内容物中大肠杆菌、乳酸菌和双歧杆菌活菌数分别为5.57士0.16(lg CFU/g)、6.32±0.09 (lg CFU/g)和5.54±0.17(lg CFU/g);各抗菌肽组的大肠杆菌(除2 mg/kg LFP-20外)均显著低于E.coli组(p<0.05),乳酸菌数和双歧杆菌数(除8 mg/kg LFP-20外)均显著高于E. coli组(p<0.05)。E.coli组粪便中大肠杆菌、乳酸菌和双歧杆菌活菌数分别为6.07±0.09 (lg CFU/g)、5.88±0.04(lg CFU/g)和5.88±0.04(lg CFU/g),各抗菌肽组粪便大肠杆菌活菌数均显著低于E.coli组(p<0.05); 2 mg/kg LF-2组的乳酸菌数显著高于E.coli组(p<0.05)；六个抗菌肽组的双歧杆菌数均显著高于E.coli组(p<0.05)。上述研究结果揭示,LFP-20及改良肽LF-2、LF-6可以通过体内抑菌作用增强小鼠抵抗E.coli K88感染能力,同时,能够改善由于感染引起的肠道双歧杆菌和乳酸菌数量降低,并且改良肽LF-2和LF-6的体内抑菌效果好于模板LFP-20。
4.猪乳铁蛋白肽及改良肽对小鼠免疫功能影响的研究
LFP-20及改良肽LF-2、LF-6对ICR小鼠免疫功能影响的研究结果表明：与正常对照组小鼠相比,2 mg/kg的LF-2和LF-6显著增加了小鼠的胸腺指数(p<0.05),而8 mg/kg的LF-2和LF-6显著降低了小鼠的胸腺指数(p<0.05)和脾脏指数(p<0.05); 8 mg/kg LF-6显著提高了外周血淋巴细胞百分率(p<0.05),2 mg/kg LF-6显著提高了小鼠外周血白细胞总数；2 mg/kg的LFP-20、LF-2、LF-6和8 mg/kg LF-2、LF-6显著降低了小鼠外周血CD3+CD4+淋巴细胞的比例,2 mg/kg LF-2、LF-6和8 mg/kg LF-2显著增加了小鼠外周血中的B细胞比例(p<0.05),但对NK细胞的数量没有显著影响；8 mg/kg LF-2和2 mg/kg LF-6组小鼠脾脏淋巴细胞的LPS刺激指数和ConA刺激指数显著升高(p<0.05)。
LFP-20及改良肽LF-2、LF-6对E.coli K88感染小鼠免疫功能影响的研究结果表明：与正常对照组相比,E.coli组小鼠的胸腺指数显著降低,2 mg/kg的LF-2、LF-6和8 mg/kg的LFP-20、LF-2均有使感染小鼠胸腺指数升高的趋势(p>0.05)。与正常对照组相比,E.coli组小鼠的外周血淋巴细胞百分率有所增加(p>0.05)；与E.coli组相比,六个抗菌肽组的外周血淋巴细胞百分率均显著降低(p<0.05)。与正常对照组相比,E.coli组小鼠的外周血CD3+CD8+细胞的比例显著降低,CD3+CD4+和CD3+CD8+细胞的比值显著升高,NK细胞的比例显著降低；2 mg/kg的LFP-20、LF-6和8 mg/kg的LFP-20、LF-2使小鼠外周血CD3+CD8+细胞的比例显著高于E.coli组(p<0.05)；与E.coli组小鼠相比,8mg/kg LF-2显著提高了小鼠外周血中NK细胞的比例(p<0.05)。E. coli组小鼠脾脏淋巴细胞的LPS刺激指数和ConA刺激指数均显著高于正常对照组(p<0.05)；六个抗菌肽组的LPS刺激指数和ConA刺激指数均显著低于E.coli组；与正常对照组相比,2 mg/kg和8 mg/kg LFP-20组小鼠脾脏淋巴细胞的LPS刺激指数显著升高(p<0.05)；2 mg/kgLFP-20组ConA刺激指数显著升高(p<0.05)。与正常对照组相比,E.coli组小鼠脾细胞的抗体生成能力没有显著变化,但2 mg/kg的三种抗菌肽和8 mg/kg的LF-2、LF-6使小鼠脾细胞的抗体生成能力显著提高(p<0.05)。与正常对照组相比,E. coli组细胞因子IL-1、IL-10、TNF-α和趋化因子MCP-1、MIP-1α基因表达水平显著升高(p<0.05)；LF-2降低了由E.coli感染导致的MCP-1、MIP-1α、IL-10基因表达水平升高(p<0.05),显著增加了IFN-γ基因表达水平(p<0.05)；与E. coli组相比,2 mg/kg LF-6显著降低由E. coli感染导致的MCP-1、MIP-1α、和IL-10基因表达水平；8 mg/kg LF-6显著降低了由E. coli感染导致的MCP-1、MIP-1α、IL-1、IL-10和TNF-α基因表达水平(p<0.05)。
5.猪乳铁蛋白肽改良肽的重组表达研究
改良抗菌肽LF-6在大肠杆菌中的重组表达。根据大肠杆菌密码子偏好性及改良肽LF-6的氨基酸序列设计引物,并通过套叠PCR成功扩增目的基因EK-LF-6及TEV-LF-6,将目的基因构建至表达载体pET32a,成功构建了大肠杆菌重组菌株BL21(DE3)pLysS-pET32a-TEV-LF-6和BL21(DE3)pLysS- pET32a- EK-LF-6,经诱导表达和SDS-PAGE分析,融合蛋白Trx-EK- LF-6及Trx-TEV- LF-6均有明显表达；Bradford法蛋白定量、凝胶条带分析及计算获得可溶性融合蛋白Trx-TEV-LF-6及Trx-EK-LF-6的表达量分别为40.60 mg/L和42.13 mg/L；两种融合蛋白经蛋白酶切割后,目标抗菌肽LF-6的理论表达量分别为5.59 mg/L和6.13 mg/L。对两种融合蛋白进行分离纯化及TEV酶和EK酶切割相应融合蛋白,结果表明TEV酶的切割效率高于EK酶。对切割产物进行冷冻干燥浓缩并使用琼脂糖孔穴扩散法检测其活性,结果表明TEV酶切割融合蛋白的产物对E.coli K88及ATCC25922具有一定抑菌活性,且对E.coli K88的抗菌活性强于对大肠杆菌ATCC25922,该结果表明经过TEV酶切割后残留在LF-6 N端的Gly对LF-6活性的影响较小。
改良抗菌肽LF-6在巴斯德毕赤酵母中的重组表达。根据酵母偏爱密码子及LF-6氨基酸序列设计引物,通过套叠PCR扩增获得目的基因后,将其构建至诱导型分泌表达载体pPICZaA,重组质粒PICZa-LF-6转化蛋白酶缺陷型酵母菌株SMD1168构建重组菌株SMD1168-pPICZaA-LF-6,经甲醇诱导重组菌株表达目标抗菌肽LF-6；取发酵上清进行Tricine-SDS-PAGE检测结果表明,重组菌株诱导6天后目的肽LF-6表达量达较高水平；对发酵液上清进行浓缩后,Bradford法测定LF-6的表达量为20 mg/L。琼脂糖孔穴扩散法对抑菌活性的检测结果表明重组表达的LF-6对E.coli K88具有一定的抑菌活性。
综上所述,本论文通过对LFP-20的分子改良,获得了对革兰氏阴性菌和革兰氏阳性菌抗菌活性明显提高且在一定浓度范围内对红细胞溶血率和外周血单核细胞毒性没有显著增强的改良肽LF-2和LF-6；与模板肽LFP-20相比,改良肽LF-2和LF-6对细菌具有更强的破膜作用机制,并可以通过体内抑菌作用增强小鼠抵抗E. coli K88感染能力,改善由于感染引起的肠道有益菌双歧杆菌和乳酸菌数量降低：改良肽LF-2和LF-6还能够通过改善小鼠胸腺指数、外周血中B细胞比例、刺激脾脏淋巴细胞转化等发挥免疫调节功能,控制由于E.coli感染导致的动物模型免疫指标异常变化：此外,还利用大肠杆菌和毕赤酵母表达系统成功表达了改良肽LF-6,理论表达量分别为5.59 mg/L和20mg/L,表达产物对E. coli具有明显抑菌活性。
Porcine lactoferricin-20 (LFP-20) is a 20-amino acid residue of cationic antimicrobial peptide derived from the porcine lactoferrin N terminus. In this study, we designed the analogs using LFP-20 as a template and screening the antibacterial activity, hemolytic activity and cytotoxicity to peripheral blood mononuclear cells of analogs. Furthermore, the comparative studies of modified peptides LF-2, LF-4, LF-6 and LFP-20 in the membrane mechanism of action were conducted. The protective effects of analogs against E.coli were investigated using mouse model with E.coli infection in thigh and abdominal cavity. Then, effects of analogs on the immune function of health and infected mouse with Escherichia.coli infection were discussed. Moreover, the analog LF-6 was expressioned successfully using E.coli expression system and Pichia pastoris expression system. The main results are as follows:
1. Molecular improvement and screening of porcine lactoferricin
Eight analogs were obtained by strategies of removing the intermolecular disulfide bonds, changing the proportion of hydrophobic and aromatic amino acids of LFP-20 based on the analysis of LFP-20 physicochemical properties, amino acid composition and structure prediction. Compared to LFP-20, the analogs LF-2, LF-4 and LF-6 exhibited 2～64 times increased antimicrobial activities against gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, Salmonella choleraesuis, Salmonella typhimurium, and 2-8 times increased antibacterial activities against gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis. Among three analogs, LF-6 showed the best antimicrobial activities. LF-2, LF-4 and LF-6 did not induce increased hemolytic activity significantly (p> 0.05) to human and porcine erythrocyte below 32μg/mL compared to LFP-20. Furthermore, the proliferations of human and porcine peripheral blood mononuclear cells (PBMCs) were not influenced significantly (p> 0.05) by LF-2, LF-4 and LF-6 below 50μg/mL. Except 8μg/mL and 16μg/mL LF-4, the cytotoxicities of LF-2, LF-4 and LF-6 to PBMCs were not also increased significantly (p> 0.05) below 32μg/mL compared to LFP-20.
2. Membrane mechanism of action of porcine lactoferricin and analogs
The effects of LFP-20 and its analogs LF-2, LF-4, LF-6 on E.coli and S.aureus morphology observed by SEM and TEM showed four peptides led to different degrees of membrane protrusions or damage, the cell wall and membrane abruption, cell contents leak, decreasing cytoplasmic electron density at 1×MIC after 30 min incubation. These results indicated that cytoplasmic membrane of bacteria is one of the important targets of LFP-20 and its analogs LF-2. LF-4, LF-6.
At the same concentration, three analogs LF-2, LF-4, LF-6 caused stronger membrane depolarization of E.coli and S.aureus than LFP-20, which more likely to lead to ion channel formation on cell membrane. Compared to LFP-20, three analogs enhanced the E.coli outer membrane permeability significantly (p<0.05) at 8～32μg/mL. Among four peptides, LF-6 led to the most rapid and obvious inner membrane permeability of E.coli. LFP-20, LF-4, LF-6 had similar inhibiting rate (Imax) to binding ability between DPX and LPS, which were 68%,56% and 51% respectively. However, the concentrations of LF-4 and LF-6 required for 50% inhibition rate (I50) to binding ability between DPX and LPS were significantly lower than LFP-20, which were 7.96μg/mL and 6.91μg/mL respectively, and the I50 of LFP-20 was 17.30μg/mL. Although the ability of replacement DPX molecule in DPX-LPS of LF-4, LF-6 weaker than LFP-20, peptides concentration required to achieve maximum binding with LPS for LF-4 and LF-6 lower significantly than LFP-20. Four antimicrobial peptides had similar damage potential to the liposome membrane at 16μg/mL and 32μg/mL. However, compared with LFP-20, three analogs presented a more damage potential on liposome membrane PC: PG (1:1) and PG at 64μg/mL, causing more than 40% calcein release. These results revealed that membrane disruption mechanism is the major mechanism of action of LFP-20 and three analogs against E.coli and S.aureus. Compared with LFP-20, the enhanced antimicrobial activity of LF-2, LF-4 and LF-6 were related to the increased cytoplasmic membrane depolarization, inner and outer membrane permeability, more easily combined with LPS and the damage potential to liposome membrane.
3. Protective effects of porcine lactoferricin and analogs on E.coli infected mouse
Based on the study of acute toxicity of LFP-20 and analogs LF-2, LF-6 to ICR mouse, the mouse thigh and abdominal infection model with E.coli K88 were established to compare the protective ability LFP-20 and LF- 2, LF-6 against E.coli infection. The results showed that LD50 of LFP-20 and LF-2, LF-6 were 34.25 mg/kg,16.54 mg/kg and 29.52 mg/kg. The effects of LFP-20 and LF-2, LF-6 to mouse thigh-infection model showed that three peptides have a protective effect on mouse infected with E.coli K.88 at 2 mg/kg and 8 mg/kg. In addition to 2 mg/kg LFP-20, another five doses of the peptides significantly reduced the number of in thigh muscle (p<0.05). The inhibitory effect of 8 mg/kg LF-6 in thigh muscle was the most significant (p<0.05), and the number of E.coli in homogenate of thigh muscle was 3.85±0.24 (lg CFU/g).
The effects of LFP-20 and LF-2, LF-6 on mouse abdominal infection model showed that 2 mg/kg and 8 mg/kg of the three peptides could inhibit the infection of E.coli K88 significantly (p<0.05) of peritoneal fluid, liver and mesenteric lymph nodes in mouse model. The inhibitory effect of 8 mg/kg LF-6 was the most significant (p<0.05) compared with E.coli control, and the number of E.coli in homogenate of peritoneal fluid, liver and mesenteric lymph nodes were 1.18±0.10 (lg CFU/mL),3.85±0.24 (lg CFU/g) and 3.00±0.15 (lg CFU/g), respectively. The number of E.coli, Lactobacillus and Bifidobacterium in cecum contents of E.coli control were 5.57±0.16 (lg CFU/g),6.32±0.09 (lg CFU/g) and 5.54±0.17 (lg CFU/g). The number of E.coli in cecum contents of peptides groups (in addition to 2 mg/kg LFP-20) were significantly lower than E.coli control (p<0.05), and the number of Lactobacillus and Bifidobacterium (in addition to 8 mg/kg LFP-20) were significantly higher than E.coli control (p<0.05). The number of E.coli in fecal of six peptides groups were significantly lower than E.coli control (p<0.05), and the number of Lactobacillus of 2 mg/kg LF-2 were significantly higher than E.coli control (p<0.05). The number of Bifidobacterium of six peptides groups were significantly higher than E.coli control (p<0.05). The results revealed that the ability to resist E.coli K88 infection of mouse could be enhanced by LFP-20 and analogs LF-2, LF-6 through bacteriostasis in vivo and improve the number decrease of Lactobacillus and Bifidobacterium caused by intestinal infection at the same time. LF-2 and LF-6 held the better bacteriostasis than LFP-20 in vivo.
4. Effects of porcine Iactoferricin and analogs on immune function in mouse
The results of effect of LFP-20 and analogs LF-2, LF-6 on immune function in ICR mice showed that 2 mg/kg LF-2 and LF-6 increased the mouse thymus index(p<.05); 8 mg/kg LF-2 and LF-6 reduced mice thymus index (p<0.05) and spleen index (p<0.05) compared with control mouse. The percentage of peripheral blood lymphocytes and the total number of peripheral white blood cells were also increased significantly (p<0.05) by 8 mg/kg LF-6 and 2 mg/kg LF-6 respectively. The proportion of peripheral blood CD3+CD4+ lymphocytes of mouse were reduced significantly (p<0.05) by LFP-20, LF-2, LF-6 at 2 mg/kg and LF-2, LF-6 at 8 mg/kg.2 mg/kg LF-2, LF-6, and 8 mg/kg LF-2 could increased the proportion of peripheral blood B cells significantly (p<0.05), but had no significant influence on the number of NK cells. The LPS stimulation index and ConA stimulation index to spleen lymphocytes in 8 mg/kg LF-2 and 2 mg/kg LF-6 group were significant (p<0.05) higher than that of control group.
The results of effect of LFP-20 and analogs LF-2, LF-6 on immune function in ICR mice infected with E.coli K88 showed that the thymus index of mouse in E.coli control was decreased significantly compared to the health control. The thymus index of 2 mg/kg LF-2, LF-6, and 8 mg/kg LFP-20, LF-2 groups had increasing trend compared to E.coli control. Compared with health control, the percentage of peripheral blood lymphocytes of E.coli control had increasing trend (p> 0.05). But the LFP-20 and analogs LF-2, LF-6 decreased the percentage of peripheral blood lymphocytes significantly (p<0.05) compared to E.coli control. The peripheral blood CD3+CD8+numbers of E.coli control was significantly decreased and the ratio of CD3+CD4+and CD3+CD8+increased significantly (p<0.05). The number of NK cells was also reduced significantly (p<0.05) by E.coli infection. The number of peripheral blood CD3+CD8+cells of 2 mg/kg of LFP-20, LF-6, and 8 mg/kg of the LFP-20, LF-2 groups were significantly higher than that of E.coli control (p<0.05), and 8 mg/kg LF-2 increased the proportion of peripheral blood NK cells significantly (p<0.05). The LPS and ConA stimulation index to mouse spleen lymphocytes of E.coli control were significantly higher than that of health control (p<0.05). Six peptides decreased the LPS and ConA stimulation index significantly (p<0.05) compared to E.coli control. The LPS stimulation index to mouse spleen lymphocytes of 2 mg/kg and 8 mg/kg LFP-20 the ConA stimulation index of 2 mg/kg LFP-20 was significantly higher (p<0.05) than E.coli control. Compared with health control, the antibody formation ability of spleen cells in mouse of E.coli control did not influenced significantly, but 2 mg/kg of three antimicrobial peptides and 8 mg/kg LF-2, LF-6 enhanced the antibody formation ability of spleen cells significantly (p<0.05). The gene expression levels of cytokines IL-1, IL-10, TNF-a and chemokine MCP-1, MIP-la of E.coli control were significantly higher than those of health control (p<0.05). LF-2 reduced the gene expression level of MCP-1 and increased the gene expression level of MIP-1α, IL-10 significantly (p<0.05) compared with E.coli control.2 mg/kg LF-6 reduced the MCP-1, MIP-la and IL-10 gene expression levels significantly (p<0.05), and 8 mg/kg LF-6 reduced the MCP-1, MIP-la, IL-1, IL-10 and TNF-a gene expression level significantly (p<0.05) compared with E.coli control.
5. The recombinant expression of porcine Iactoferricin analog
The recombinant expression of analog LF-6 in E.coli. The primers were designed according to E.coli codon preference and amino acid sequence of LF-6, then target genes EK-LF-6 and TEV-LF-6 were amplified successfully by overlap PCR. After constructed the target genes into expression vector pET32a, the recombinant E. coli strain BL21 (DE3) pLysS- pET32a- TEV- LF-6 and BL21 (DE3) pLysS- pET32a- EK- LF-6 were obtained. The expression of fusion protein Trx-EK-LF-6 and Trx-TEV-LF-6 were detected through IPTG induction and SDS-PAGE analysis. Expression levels of soluble fusion protein Trx-TEV-LF-6 and Trx-EK-LF-6 were 40.60 mg/L and 42.13 mg/L respectively by proteins determination using Bradford method, gel analysis and calculated. The theory expression levels of LF-6 were 5.59 mg/L and 6.13 mg/L after two fusion proteins cleaved by protease. The two fusion proteins cleaved by TEV and EK after purification. Results showed that the cleavage efficiency of TEV higher than EK. The antimicrobial activies of cleavage products were detected using agar hole diffusion method after freeze-drying of the products. Results showed that cleavage products of TEV had antimicrobial activities against both E.coli ATCC25922 and E.coli K88, but exhibited higher potential to against E.coli K88. These indicated that the Gly residue in the LF-6 N terminal less affected the antimicrobial activity of LF-6 after TEV cleavage
The recombinant expression of analog LF-6 in P. pastoris. The primers were designed according to yeast codon preference and amino acid sequence of LF-6, then target gene were amplified successfully by overlap PCR. After constructed the target gene into expression vector pPICZaA, recombinant plasmid PICZa-LF-6 were transformed into protease-deficient yeast strain SMD1168. Recombinant strain P.pastoris SMD1168-pPICZaA-LF-6 was obtained. The expression of antimicrobial peptides LF-6 was induced by methanol and the fermentation supernatant were detected. Tricine-SDS-PAGE results showed that the expression level of target peptide LF-6 reached a high level after the recombinant strain induced for 6 days. LF-6 expression level was measured using the concentrated fermentation supernatant by Bradford method, which was 20 mg/L. The expression product exhibited the antimicrobial activities against E.coli K88
In summary, we obstained the analogs LF-2 and LF-6 with increased antimicrobial activity against gram-negative and gram-positive bacteria through the molecular improvement of LFP-20. The hemolysis rate and cytotoxicity to PBMCs of LF-2 and LF-6 were not enhanced significantly at a certain range of concentration. Compared to template peptide LFP-20, analogs LF-2 and LF-6 showed stronger damage potential on bacterial cytoplasmic membrane and enhanced capacity to resist the infection of mice with E.coli K88 in vivo. LF-2 and LF-6 could also improve the number reduction of Bifidobacterium and Lactobacillus caused by infection. LF-2 and LF-6 were able to regulate the immune functions through changing the thymus index, proportion of B cells in peripheral blood, transformation efficiency of spleen lymphocyte, and control the abnormal changes in immune parameters ofanimal model caused by E.coli infection. In addition, analog LF-6 were expressed successfully using E. coli and P. pastoris expression system. The theory expression levels of LF-6 were 5.59 mg/L and 20mg/L repectively, and products exhibited obvious antibacterial activity against E.coli.
引文
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