Recombinant lactobacillus of insulin-like growth factor and application thereof

A technology for insulin-like growth factors, applied in the direction of insulin-like growth factors, applications, recombinant DNA technology, etc., can solve the problems of low protein activity and low expression, achieve good economic benefits, promote intestinal development, and reduce production costs Effect

Active Publication Date: 2014-03-12
ZONHON BIOPHARMA INST +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the inventors tried to express porcine IGF through experiments by directly transferring porcine IGF gene into recombinant lactic acid bacteria, but due to species differe...

Method used

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  • Recombinant lactobacillus of insulin-like growth factor and application thereof
  • Recombinant lactobacillus of insulin-like growth factor and application thereof
  • Recombinant lactobacillus of insulin-like growth factor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] 实施例1重组猪胰岛素样生长因子的基因优化设计

[0040] 发明人根据GenBank已公开的猪胰岛素样生长因子(insulin-like growth factor1,IGF1)的cDNA序列(GenBank登录号:NP_999421.1),对该基因进行密码子优化后得到本发明的重组猪胰岛素样生长因子基因,如SEQ ID No:1所示。下面是对猪胰岛素样生长因子基因进行密码子优化,优化前后各参数对比如下:

[0041] 1.密码子适应指数(Codon Adaptation Index,CAI)

[0042] Depend on Figure 2-a 可知,密码子没有优化前,猪胰岛素样生长因子基因在乳酸菌中密码子适应指数(CAI)为0.37。 Depend on Figure 2-b 可知,通过密码子优化后,使得本发明的猪胰岛素样生长因子基因在乳酸菌中CAI指数为0.92。通常CAI=1时被认为该基因在该表达系统中是最理想的高效表达状态,CAI指数越低表明该基因在该宿主中表达水平越差,因此可以看出经过了密码子优化后得到的基因序列可以提高猪胰岛素样生长因子基因在乳酸菌中的表达水平。

[0043] 2.最优密码子使用频率(Frequency of Optimal Codons,FOP)

[0044] Depend on Figure 3-a 可知,基于乳酸菌表达载体,密码子没有优化前,猪胰岛素样生长因子基因序列的低利用率密码子出现百分比为73%。 这条未进行优化的基因含有串联稀有密码子,这些密码子可能降低翻译效率,甚至能够解散翻译装配物。 Depend on Figure 3-b 可知,通过密码子优化后,本发明的猪胰岛素样生长因子基因在乳酸菌系统中出现低利用率密码子的频率为1%。

[0045] 3.GC碱基含量(GC curve)

[0046] GC含量理想分布区域为30%-70%,在这个区域外的出现任何峰都会不同程度地影响转录和翻译效率。 Depend on Figure 4-a , Figure 4-b 的猪胰岛素样生长因子基因的GC碱基平均含量分布区域图对比可知,由 Figure 4-a 中显示猪胰岛素样生长因子基因中在优化前GC碱基平均含量为64.16%,由 Fi...

Embodiment 2

[0053] 实施例2重组猪胰岛素样生长因子基因的表达质粒构建

[0054] 将优化后的重组猪胰岛素样生长因子全基因(如SEQ ID No:1所示)合成的片段,构建到pUC57质粒(购自南京金斯瑞科技有限公司)中,得到一种长期保存质粒,记为pUC57-poIGF质粒。以pUC57-poIGF质粒为模板,上、下游引物分别引入Nsi I和Xho I酶切位点,进行PCR扩增,所用引物序列如下:

[0055] Upstream primers:

[0056] P1:GCCATGCATATTTTAATAAACCAA

[0057] 下游引物:

[0058] P2:GTTCTCGAGTTAAGTATTTTTAAGA

[0059] 反应总体积50μL,其中浓度为10μmol / L引物各加2.5μL,浓度为10mmol / L的dNTP加1μL,所用DNA聚合酶Phusion High-Fidelity DNA polymerase(购自Theromo-Fisher scientific),2U / μL,加0.5μL。 反应条件为98℃5s、56℃30s、72℃15s,25个循环后,产物经1.0%琼脂糖凝胶电泳分析,结果显示产物大小与预期大小(210bp)一致。 (Such as Figure 7 所示)

[0060] 将得到的基因产物用DNA凝胶回收试剂盒(购自北京天根生化科技有限公司)纯化。纯化后,用Nsi I和Xho I(购自New England Biolabs公司)双酶切,用T4连接酶(购自New England Biolabs公司)连接到PCYT质粒(购自中国质粒载体菌株细胞株基因保藏中心)中,转化到大肠杆菌DH5α感受态细胞(购自北京天根生化科技有限公司)中,在含有25μg / mL的氯霉素(购自Amersco公司)的LB固体培养基中37℃培养过夜。第二天筛选阳性克隆菌测序,提取质粒,酶切后电泳鉴定目标条带大小正确(如 Figure 8 shown). 经测序,与预期序列完全一致,即得到重组猪胰岛素样生长因子的表达质粒,记为PCYT-poIGF。

Embodiment 3

[0061] 实施例3乳酸乳球菌感受态的制备及转化

[0062] 1、乳酸乳球菌感受态细胞的制备

[0063] 将贮存L.lactics NZ9000甘油菌(购自中国质粒载体菌株细胞株基因保藏中心)接种于10mL GM17培养基(购自青岛海博生物),30℃过夜静置培养。将1mL过夜培养物接种于50mL含2.5%甘氨酸的SGM17培养基(购自青岛海博生物)中,30℃静置培养至OD 600 值为0.5左右,冰浴,用OSPS缓冲液(0.5M蔗糖、10%甘油)洗涤菌体,再用OSPS缓冲液重悬细胞,-70℃保存或直接使用。

[0064] 2、乳酸乳球菌的电转化

[0065] 将质粒PCYT-poIGF和乳酸乳球菌感受态细胞在电转化杯中混合,使用2500V(电容25μ,电阻200Ω)脉冲输出,电转化乳酸乳球菌。将转化后的菌体,加入预冷的SMG17MC(GM17+20mM CaCl 2 +2mM MgCl 2 ),30℃静置孵育2h。洗涤细胞,涂布含氯霉素(7.5mg / mL)的GM17琼脂糖固体培养基。

[0066] 转化后涂布的平板上出现抗氯霉素的阳性克隆。将重组单菌落接种于含有氯霉素(终浓度7.5μg / mL)的液体GM17培养基中,30℃恒温静置培养过夜,再按1%的接种量转接到5mL新鲜的含氯霉素(终浓度7.5μg / mL)的液体GM17培养基中,Nisin(购自Sigma)诱导检测猪胰岛素样生长因子的表达情况。

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Abstract

The invention provides an encoding gene of a recombinant insulin-like growth factor, lactobacillus containing the encoding gene and application of the lactobacillus, and belongs to the field of bio-genetic engineering. The insulin-like growth factor is a multifunctional cell proliferation regulation factor, has important acceleration in differentiation, proliferation, and growth and development of cells, but has the problems of insufficient throughout, high price, required purification of product, difficult drug administration and the like just as most of genetic engineering drugs. In order to obtain a lot of recombinant insulin-like growth factors, recombinant insulin-like growth factor genes optimized by codons are subjected to heterologous expression by adopting a lactobacillus expression system, and the recombinant lactobacillus is utilized as a living bacteria agent or a feed additive to enhance the nonspecific immunity of a pig.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant lactic acid bacterium containing insulin-like growth factor, and an expression method and application of the coding gene of the insulin-like growth factor and its protein in the recombinant lactic acid bacterium. Background technique [0002] The animal intestine is not only the largest digestive and absorption organ in the animal body, the final absorption site of all nutrients, but also the largest immune organ in the body. The healthy development of piglet intestinal tract is the physiological basis for rapid growth and high survival rate. Due to the rapid development of the intestinal tract of newborn piglets, the structure and function are not perfect, the digestion and absorption of nutrients and the immune barrier function have great limitations, and it is difficult to adapt to changes in nutrition and environment, which increases the difficulty of feeding. Durin...

Claims

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Application Information

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IPC IPC(8): C12N15/16C12N15/63C12N1/21C07K14/65A23L1/29A23K1/16C12R1/46A23L33/00
Inventor 马永侯景罗成章成昌姚翔徐春林陈晨王耀方
Owner ZONHON BIOPHARMA INST
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