Symbiotic system for promoting growth of chicks through cooperation of Klotho protein and lactic acid bacteria

Abstract

The invention develops a Klotho protein delivery system based on recombinant lactic acid bacteria. The Klotho protein delivery system is characterized in that optimized Klotho genes are integrated into a food-grade carrier pNZ8149 through genetic engineering, and the food-grade carrier pNZ8149 is introduced into lactococcus lactis NZ3900 to construct a recombinant strain for expressing Klotho protein; and the functional Klotho protein can be efficiently secreted through Nisin induction. The preparation is orally taken in the form of viable bacteria to feed chicks, targeted release of Klotho protein is realized through intestinal colonization, and experiments prove that the preparation can synergistically activate growth and metabolism pathways (the weight of 21-day-old chicks is increased, and the feed conversion ratio is reduced) and improve intestinal health. The technology breaks through the limitation that traditional probiotics are lack of functional protein delivery capacity, an antibiotic / hormone growth promoter is replaced by a green scheme without resistance and zero residue, and a new safe and efficient growth regulation strategy is provided for livestock and poultry breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and animal nutrition, and specifically relates to a method for expressing Klotho protein by genetically engineering Lactococcus lactis (Lactococcus lactis NZ3900), and application of the recombinant probiotic preparation in promoting growth of chicks and improving intestinal health. BACKGROUND

[0002] Lactococcus lactis is a microorganism recognized as safe worldwide and has multiple probiotic effects, and is often used for genetic engineering. As a representative strain of lactic acid bacteria, it is considered safe by the FDA and can be used in food fermentation, drug production, feed additive, etc. It grows fast, is easy to operate, and has a clear genetic background, making it an ideal host strain for genetic engineering. In recent years, with the development of molecular biology, it has been widely used as a carrier for presenting viral and bacterial antigens. In contrast, traditional antibiotic / hormone growth promoters are banned due to drug resistance, residues and ecological risks, and conventional probiotics cannot target the delivery of growth-promoting functional proteins, and existing recombinant expression systems have many defects, and engineered probiotics often have hidden risks of residual drug resistance genes, which restrict their application, but Lactococcus lactis has outstanding advantages and broad application prospects.

[0003] Klotho is a transmembrane protein expressed in endocrine tissues such as kidney, choroid plexus and anterior pituitary. It was first found to have anti-aging effects and can act as an FGF23 cofactor to regulate phosphate homeostasis and inhibit insulin and IGF-1 signaling pathways. It can also form a circulating hormone through an ADAM protease-mediated shedding process that can be activated by insulin. It is highly expressed in the anterior pituitary and acts as a positive regulator of growth hormone (GH) secretion, regulating IGF-1 and bFGF activity in the pituitary. Deficient mice have smaller body size due to reduced GH secretion granules in the pituitary, indicating that the IGF-1 and bFGF pathways may be mediators of GH secretion. GH significantly upregulates Klotho expression after increasing IGF1 levels. Clinical studies have shown that serum Klotho levels increase in patients with GH deficiency (GHD) and chronic kidney disease (CKD) after GH treatment and are positively correlated with IGF1. The reduction of Klotho is also closely related to cardiovascular risk, vascular calcification and arterial stiffness. Its protective mechanism involves the Klotho-FGF23 system inducing iNOS expression in vascular endothelial cells to promote NO production, and FGF23 enhancing the biological effects of Klotho. Klotho not only promotes growth hormone secretion by regulating the IGF-1 and bFGF pathways, but also maintains vascular tone in the cardiovascular system in cooperation with FGF23. Its multiple roles in the endocrine and cardiovascular systems collectively reflect its role in promoting growth and overall health of the body.

[0004] Prior art problems: Traditional growth promoters such as antibiotics, hormone additives are easy to cause drug resistance, drug residues and ecological risks, and have been banned in many countries. Although conventional probiotics (such as lactic acid bacteria) can regulate intestinal flora, they lack the ability to target the delivery of specific growth-promoting functional proteins. Klotho protein is a growth-promoting factor with functions of promoting growth, enhancing antioxidant and regulating metabolism, but there is no technical solution for delivering Klotho protein through probiotics at present.

[0005] Innovation and advantages: For the first time, Klotho protein is combined with probiotic delivery technology to achieve the synergistic effect of growth promotion and maintenance of intestinal homeostasis. The GC content of the gene sequence of the first 958 bp of Klotho is optimized to 70%. According to the principle of amino acid invariability, the synthesis success rate and protein expression success rate of the sequence are improved based on the parameters of GC content, CIS element, repeat element, RNA splicing site, ribosome binding sequence, and minimum free energy of mRNA. Food-grade carrier and strain NZ3900 / pNZ8149 ensure no risk of drug-resistant gene residues. Cost-effective: protein purification process is free, which can be directly produced on a large scale through fermentation, reducing application cost. SUMMARY

[0006] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a food-grade lactic acid bacterial strain secreting antibiotic-resistant secretory growth-promoting secretory Klotho protein.

[0007] Another purpose of the present application is to provide a gene encoding the growth-promoting secretory Klotho protein after optimization.

[0008] Still another purpose of the present application is to provide a recombinant expression vector pNZ8149-Kloho containing a gene encoding growth-promoting secretory Klotho protein.

[0009] Still another purpose of the present application is to provide the application of the gene of the growth-promoting secretory Klotho protein, the recombinant expression vector and the recombinant bacteria expressing Klotho protein.

[0010]

[0011] A recombinant expression vector containing the gene encoding the growth-promoting secretory Klotho protein.

[0012] A method for constructing the recombinant expression vector, comprising the following steps: The gene sequence of Klotho-3xFLAG is designed, that is, 3xFLAG sequences are added at the ends of the Klotho gene sequence, and homologous arm sequences matching the pNZ8149 vector sequence are added to the two sections of the Klotho-3xFLAG gene sequence. After the designed gene sequence is synthesized by a company, it is assembled into pNZ8149 by homologous recombination to obtain the recombinant vector pNZ8149-Klotho-3xFLAG.

[0013] The purification is preferably performed using a conventional plasmid purification kit.

[0014] The method for constructing the recombinant expression vector further comprises a step of verifying the pNZ8149-Klotho plasmid obtained in step (2); specifically, the pNZ8149-Klotho plasmid is transformed into NZ3900 competent cells, positive clones are screened using lactose as a single carbon source and plasmid amplification is performed, positive colonies are picked for enzyme digestion identification and sequencing, and the pNZ8149-Klotho plasmid is obtained when the enzyme digestion identification and sequencing results are correct.

[0015] The PCR amplification system in step (3) is as follows: PCR amplification system: PrimeSTAR Max Premix (2X) 25 μL, primer F1 1 μL, primer R1 1 μL, template 2 μL, ddH2O 21 μL.

[0016] The PCR reaction conditions in step (3) are as follows: 98℃, 10s; 57℃, 30s; 72℃, 60s; 35 cycles.

[0017] A recombinant bacterium expressing Klotho protein, which is obtained by transforming the recombinant expression vector into Lactococcus lactis.

[0018] The Lactococcus lactis is preferably Lactococcus lactis NZ3900.

[0019] A method for preparing Klotho protein, comprising the following steps: transforming the recombinant bacterium expressing Klotho protein into Lactococcus lactis NZ3900 competent cells, culturing, adding Nisin for inducing expression, centrifuging to collect bacterial cells, crushing, separating and purifying to obtain Klotho protein; wherein the conditions for inducing expression are as follows: induction temperature 30℃, induction time 5-10h, and Nisin final concentration 25-50ng / ml.

[0020] The inducing expression condition is preferably an inducing temperature of 30 DEG C, an inducing time of 3h, and a final concentration of Nisin of 50ng / ml.

[0021] The application of at least one of the growth-promoting secretory Klotho protein, the gene encoding the growth-promoting secretory Klotho protein, the recombinant expression vector, and the recombinant bacteria expressing the Klotho protein in preparing a growth-promoting lactic acid bacterial preparation.

[0022] The present application has the following advantages and effects relative to the prior art: the present application adopts genetic engineering technology and strategy, optimizes the codon sequence of the target gene, and adds a 3xFLAG tag to the end of the recombinant gene, so that the gene base sequence and the target protein are easier to monitor, optimize and screen a strain of recombinant growth-promoting protein expressed by a prokaryotic expression vector.

[0023] The present application constructs a prokaryotic vector expression system, and further detection by a chick growth promotion experiment shows that the purified protein has good growth-promoting function.

[0024] The recombinant lactic acid bacterial strain stably expressing the Klotho protein obtained in the present application is expected to be beneficial to its application in industrialized production and in anti-aging, regulation of nitric oxide generation, anti-oxidative stress, regulation of calcium and phosphorus metabolism, anti-inflammation, and cancer inhibition.

[0025] (1) The method for producing the target protein by genetic engineering is feasible, has low production cost, and is easy to mass-produce industrially; (2) The recombinant Klotho protein shows good growth-promoting activity in in vitro cell activity detection; (3) The technical target solves the safety problem of traditional growth promoters, provides a green solution for delivering Klotho protein by recombinant lactic acid bacteria, and realizes the dual improvement of the growth performance and health level of chicks. BRIEF DESCRIPTION OF DRAWINGS Figure 1 Nucleic acid electrophoretogram of Klotho-3xFLAG gene PCR identification. Figure 2 Colony map of recombinant expression vector pNZ8149-Klotho transformed into NZ3900. Figure 3 Plasmid sequencing map of recombinant expression vector pNZ8149-Klotho transformed into NZ3900 positive colony. Figure 4 Western Blot detection result of Klotho protein. Figure 5 Chick growth curve graph. Figure 6 Chick feed-meat ratio. Figure 7 : Intestinal morphology analysis of chicks (HE staining). Figure 8 : Intestinal crypt depth of chicks. Figure 9 : Intestinal villus-crypt ratio of chicks. Figure 10 : Expression levels of intestinal inflammatory factors IL-6 and TNF-α of chicks. Technical solutions

[0026] Klotho gene acquisition Gene acquisition and primer design: Obtain human Klotho protein gene (GenBank accession number: XM_006719895.3) from NCBI, and design specific primers containing 3×FLAG tag: Upstream primer: 5'-AAGGAGGCACTCACCATGGGCATG[Klotho start sequence]-3' Downstream primer: 5'-GAGCTCTCTAGAGGTACCttaCTTGTCA[3×FLAG tag termination sequence]-3'.

[0027] Expression vector construction: Amplify the Klotho+3×FLAG gene fragment by PCR, verify by nucleic acid electrophoresis, and clone into the pNZ8149 vector (containing Nisin inducible promoter). Electroporation and screening: The recombinant vector is electroporated into the NZ3900 strain, and positive clones are screened on Elliker plates, and the correctness of the gene is verified by sequencing.

[0028] Induction expression conditions: Nisin concentration (20-50 ng / mL), temperature (30°C), time (5-10 hours), and Klotho protein expression is verified by SDS-PAGE and Western Blot (anti-FLAG antibody) (Figure 1).

[0029] Recombinant bacteria fermentation culture: Positive strains are picked into GM17 medium and cultured overnight at 30°C. The next day, the overnight bacterial solution is diluted by 2% into fresh GM17, and cultured to OD600=0.4. Nisin inducer with a final concentration of 50 ng / ml is added, and the bacteria are collected by centrifugation after 3 hours of induction, and resuspended in physiological saline to 10^9 CFU / mL.

[0030] Application method

[0031] Feeding scheme: 4-5 day-old chicks are continuously fed with recombinant bacteria preparation (10^9 CFU / bird / day), and the control group is physiological saline.

[0032] Effect evaluation: determine weight gain, feed conversion, intestinal morphology (villus height / crypt depth), intestinal inflammatory factor IL-6 and tumor necrosis factor alpha expression. DETAILED DESCRIPTION

[0033] Example 1: Construction and expression verification of recombinant strain 1. Optimize Klotho gene sequence and add 3xFLAG tag, amplify Klotho gene by PCR ( Figure 1 ), and connect to pNZ8149 vector ( Figure 2 ); 2. pNZ8149-Klotho expression vector is electroporated into NZ3900 strain ( Figure 3 ), positive clones are screened and sequenced ( Figure 4 ); 3. Extract the positive strain of bacterial protein, Western Blot shows that Klotho protein (about 70 kDa) is successfully expressed ( Figure 5 ).

[0034] Example 2: Growth promotion experiment of chicks Group design: experimental group: 4-5 day-old chicks are continuously fed with recombinant bacterial preparation (10^9 CFU / each / day) for 21 days, control group: 4-5 day-old chicks are continuously fed with physiological saline for 21 days. Recombinant bacterial preparation (10^9 CFU / each / day) is fed. The body weight of chicks is weighed every 7 days, the daily feed intake is weighed, and the intestinal HE section of chicks after 21 days of feeding is detected after 21 days of feeding. The intestinal villus length and intestinal crypt depth are measured, and the intestinal villus-crypt ratio is calculated. Intestinal RNA is extracted, and the expression of intestinal inflammatory factors and tumor necrosis factor alpha is detected by fluorescent quantitative PCR.

[0035] Results: weight gain: the body weight of the experimental group at 21 days of age is increased by ( Figure 5 ) compared with the control group; the feed conversion ratio is decreased, and the feed conversion ratio FCR of the experimental group is decreased ( Figure 6 ); obvious changes are found in the intestinal HE section of chicks ( Figure 7 ), the intestinal crypt of the experimental group is deeper ( Figure 8 ), and the villus-crypt ratio is higher than that of the control group ( Figure 9 ). Compared with the intestinal safety, the intestinal indicators IL-6 and TNF-alpha are within the normal range, and there is no toxic reaction ( Figure 10 ).

[0036] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A recombinant lactic acid bacteria secreting Klotho protein without resistance to growth-promoting, characterized by: The recombinant lactic acid bacteria carries a recombinant vector pNZ8149 containing a Klotho gene, and expresses Klotho protein by Nisin induction. 2.The recombinant bacteria expressing Klotho protein according to claim 1, characterized in that: The lactic acid bacteria is Lactococcus lactis NZ3900.

3. The gene encoding the growth-promoting secretory Klotho protein according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

1.

4. The recombinant lactic acid bacteria of claim 1 or 2, which expresses Klotho protein by Nisin induction, and the optimal induction expression conditions are: induction temperature 30℃, induction time 5-10h, and Nisin concentration 50ng / ml.

5. The method of constructing a recombinant lactic acid bacterium according to claim 1 or 2 or 3, characterized in that, The method comprises the following steps: (1) Design the gene sequence of Klotho-3xFLAG, that is, add 3xFLAG sequence at the end of the Klotho gene sequence, and add homologous arm sequences matching the pNZ8149 vector sequence to the two sections of the Klotho-3xFLAG gene sequence. After the designed gene sequence is synthesized by a company, it is assembled into pNZ8149 by homologous recombination to obtain a recombinant vector pNZ8149-Klotho-3xFLAG; (2) The recombinant vector pNZ8149-Klotho-3xFLAG is transformed into NZ3900 competent cells by click transformation, and is cultured in GM17 medium to obtain recombinant lactic acid bacteria expressing Klotho.

6. The recombinant lactic acid bacteria preparation of claim 1 or 3, wherein the optimal concentration for promoting the growth of chicks is 10^9 CFU / mL.

7. The recombinant bacteria of claim 1 or 3 or 6, wherein the promotion of the growth of chicks comprises promoting the weight gain of chicks, reducing the feed-meat ratio, and improving intestinal health.

8. The recombinant bacteria of claim 1 or 6, wherein the Klotho is a conservative sequence and is suitable for multiple species, and the multiple species include chicks, ducklings, young livestock, etc.

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