Microbe transformant for weight loss and lipid reduction, the method for constructing the transformant, and application thereof

Abstract

It discloses a transformant for weight loss and lipid reduction, which is obtained by recombining and substituting human oxyntomodulin gene into thymidylate synthase gene of L.lactis genome. Wherein the recombinant substitution is homologous recombination by artificially synthesizing gene sequences, making the sequence flanking the human oxyntomodulin gene derived from the homologous sequences of thymidylate synthase gene in the L.lactis genome, then the gene fragments are electroporated into L. lactis, to carry out homologous recombination.

Claims

1. A transformant for weight loss and blood lipid reduction, wherein the transformant is obtained by replacing Lactococcus lactis (L. lactis) thyA gene with a codon-optimized human oxyntomodulin gene through homologous recombination.

2. The transformant for weight loss and blood lipid reduction according to claim 1, wherein the nucleotide sequence of the codon-optimized human oxyntomodulin gene has a DNA sequence shown in SEQ ID NO: 1.

3. The transformant for weight loss and blood lipid reduction according to claim 1, wherein the L.lactis is one strain of L.lactis selected from the group consisting of L. lactis NZ9000, L. lactis NZ3900 and L. lactis MG1363.

4. A method of constructing a transformant for weight loss and blood lipid reduction, comprising the following steps: (1) ligate the upstream sequence of L. lactis thyA gene, the codon-optimized human oxyntomodulin gene and the downstream sequence of L. lactis thyA gene into a recombinant gene fragment; (2) electro-transform the recombinant gene fragment obtained in step (1) into L.lactis for homologous recombination, to screen a thyA.sup. OXM.sup.+ bacteria resulting in the transformant.

5. A process for treating a human disease with the transformant of claim 1, comprising a step administering the transformant prepared as drug or food to a patient in need of treatment; wherein the human disease is obesity, diabetes or fatty liver.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the results of PCR genotyping.

(2) FIG. 2 shows immunoblotting analysis of OXM expression in L.lactis.

(3) FIG. 3 shows the concentration of OXM in serum 5 hours after L.lactis feeding.

(4) FIG. 4 shows the curve of body weight change after feeding obese mice with L.lactis.

(5) FIG. 5 shows the curve of food intakes 6 weeks after feeding obese mice with L.lactis.

(6) FIG. 6 shows the effect of L.lactis in comparison with Bifidobacterium.

(7) FIG. 7 shows the CT scanning of the abdomen after feeding obese mice with L.lactis for 6 weeks. A shows the normal mice; B shows obese mice fed with NZ9000; C shows obese mice fed with NZ9000-OXM (the light grey area within the white line represents the fat area).

(8) FIG. 8 shows the weight of body fats in obese mice after fed with L.lactis for 6 weeks.

(9) FIG. 9 shows the fasting blood glucose concentration in obese mice after fed with L.lactis for 6 weeks.

(10) FIG. 10 shows the serum cholesterol level in obese mice fed with L.lactis for 6 weeks.

(11) FIG. 11 shows the curve of body weight change in diabetic mice fed with L.lactis for 5 weeks.

(12) FIG. 12 shows the cholesterol level in serum of diabetic mice fed with L.lactis for 5 weeks.

DETAILED DESCRIPTION OF THE INVENTION

(13) The present invention can be better understood from the following examples. However, it will be readily understood by those skilled in the art that the embodiments described are intended to be illustrative of the invention, not and should not be construed as limiting the invention as set forth in the claims.

Example 1: Preparation of OXM Gene Transformed L.lactis

(14) (1) Construction of Homologous Recombination Fragment:

(15) According to the issued sequence of the genome of L. lactis, the sequence of 1000 bp upstream of the thyA gene start codon and 1000 bp downstream of the termination codon are found respectively.

(16) The OXM nucleic acid sequences are optimized according to the human OXM amino acid and nucleic acid sequences, and with reference to codon usage frequencies of L.lactis. The resulting sequence is shown in SEQ ID NO: 1.

(17) Chemical synthesis of gene fragment OXM-M: the sequence comprise of 1000 bp upstream of L.lactis thyA start codon, human OXM gene sequence, and 1000 bp downstream of L.lactis thyA gene termination codon.

(18) (2) Preparation and Selection of Transgenic L.lactis Expressing OXM

(19) Electro-transform human oxyntomodulin fragment OXM-M to the strain L.lactis NZ9000, named as L. lactis OXM.

(20) Add 2 l of OXM-M to 40 l of L.lactis competent bacterial, mix well, place on the ice for 5 min, then transfer to a 0.4 cm electroporation cuvette.

(21) Place the electroporation cuvette in the shocking chamber of the electroporation apparatus, and charge the capacitor. the condition was set to voltage 2000V, capacitance 25 F, resistance 200, and pulse length 4 msec.

(22) After electroporation, remove the cuvette quickly, add to a 1 ml M17 culture medium, then place on ice for 5 min, and then incubate for 1 h at 30 C., then spread on a solid medium containing deoxythymidine and culture overnight. The next day, pick single colonies to inoculate them to the deoxythymidine-deficient and deoxythymidine-containing solid medium respectively; if strains grow in a deoxythymidine-containing solid medium but do not grow in a deoxythymidine-deficient solid medium, then the strains are positive bacteria.

(23) (3). Genotyping of the Transformants Expressing OXM

(24) Three pairs of PCR primers for genotyping are designed.

(25) Primer 1: 1F (SEQ ID No: 2) 5-GGTTTTATTGTTTCATTAGT-3, located at thyA upstream sequence;

(26) 1R (SEQ ID No: 3) 5-GAGATAATCTTTTTTTTCAT-3, located at the beginning of the OXM gene sequence.

(27) Primer 2: 2F (SEQ ID No: 4) 5-GGAATAACATTGCCTAATGA-3, located at the end of the OXM gene sequence;

(28) 2R (SEQ ID No: 5) 5-TTTATTATTAGGGAAAGCAA-3, located at thyA downstream sequence.

(29) Primer 3: 3F (SEQ ID No: 6) 5-ATGACTTACGCAGATCAAGT-3, located at the beginning of the thyA gene sequence;

(30) 3R (SEQ ID No: 7) 5-TTAAATTGCTAAATCAAATT-3, located at the end of the thyA gene sequence.

(31) 2 ul of bacterial cultures is used for standard PCR reaction.

(32) PCR products are subjected to agarose gel electrophoresis. Results are shown in FIG. 1. The strain NZ9000 is not subjected to gene recombination, only the sequence of thyA is amplified by primer 3. Since the recombinant strain NZ9000-OXM lacks the thyA gene, there is no product for PCR amplified by primer 3, while fragments containing the OXM gene sequence are amplified by primer 1 and primer 2 respectively.

(33) (4) Identification of OXM Expression

(34) Inoculate 5 ml of culture medium and incubate overnight at 30 C.

(35) On the next day, centrifuge the bacteria solution, add volume of 100% trichloroacetic acid (TCA) to the supernatant and mix well, place on the ice for 20-30 minutes, centrifuge 10 min at 12000 rpm to precipitate the protein, then discard the supernatant; add 3 volumes (of the original sample volume) of acetone. Let the samples stand at room temp for about 10 min to allow the TCA to dissolve in the acetone, then centrifuge 10 min at 12000 rpm to precipitate the protein, then discard the supernatant; add 1SDS loading buffer to dissolve the protein. Add lysozyme to the bacteria pellets, incubate for 30 min at 37 C. Mix well and add 500 l of RIPA lysis buffer for ultrasonic extraction on the ice, then centrifuge 10 min at 12000 rpm to discard the pellets, add 1SDS loading buffer to dissolve the protein.

(36) After boiled at 95 C. for 5 min, all samples are separated by 18% SDS-PAGE electrophoresis, then transferred to PVDF membrane at constant voltage under ice bath condition, after blocked with BSA at room temperature for 1 h, rinsed with TBST for 35 min, then incubated with the rabbit anti-OXM antibody (1:500) overnight at 4 C. Rinsed with TBST for 3 times, 5 min each time, HRP labeled anti-rabbit secondary antibody (1:5000) was added, incubated 1 h at room temperature, rinse with TBST for 3 times, 5 min each time, and then developed using the enhanced chemiluminescent (ECL).

(37) Results are shown in FIG. 2. The expression of OXM is not detected in L. lactis NZ9000 by immunoblotting, while the expressions of OXM are detected in the L.lactis NZ9000-OXM strain cells and the supernatant of culture medium, suggesting that the OXM is expressed in L.lactis NZ9000-OXM successfully and secreted extracellularly.

Example 2: Pharmacodynamics Experiments of OXM-Transformed Lactic Acid Bacteria to Weight Loss and Lipid Reduction

(38) (1) Prepare several weaned-stage B6 mice, and feed them high-fat diets until the obese mice models are constructed successfully. The Obesity criteria: (body weight of mice in the experimental groupaverage body weight of normal mice)/average body weight of normal mice >20%. Obese mice are divided into two groups, 10 mice in each group, and animals are fed high-fat diets continuously. One group is the control group, fed with L.lactis NZ9000; another group is the experiment group, fed with L.lactis NZ9000-OXM. At the same time, the normal control group is B6 mice fed normal diets without feeding with L.lactis.

(39) (2) Each mouse is received 510.sup.9 CFU L.lactis by intragastric administration once every 2 days. The body weights, food intakes and other vital signs of mice are observed. Five hours after the first intragastric administration, the concentration of OXM in serum was detected by mass spectrometry. Results are shown in FIG. 3, after intragastric administration of NZ9000-OXM, the serum OXM content in mice was significantly increased nearly 2.5 times, about 497 ng/ml. The result further demonstrates that L. lactis NZ9000-OXM secrete and express OXM in mice. The comparative experiments show that, when feeding equal amount of OXM recombinant Bifidobacterium (patent No. 200910041386.X), the serum OXM concentration in mice is about 380 ng/ml, which is lower than that of OXM recombinant L.lactis. The body weight monitoring data are shown in FIG. 4. After obese mice are fed L.lactis NZ9000-OXM for 4 weeks, their body weights are significantly decreased. At the 5.sup.th week, their body weights are close to normal mice. Until the end of 9-week experiment, their body weight remains at about 24 g, while the body weight of mice fed with L. lactis NZ9000 is slightly reduced. Compared with the OXM recombinant Bifidobacterium, which has been granted patent right (200910041386.X), the L.lactis OXM can reduce the body weights of obese mice by 31.1%, while the OXM recombinant Bifidobacterium can reduce the body weight by 21.4% (FIG. 6) in equal time. Thus, L. lactis NZ9000-OXM achieves better weight loss.

(40) (3) After 6 weeks of continuous feeding of L.lactis, the food intakes of mice are detected in metabolic cages for 24 h. Results were shown in FIG. 5. The food intakes of mice fed with L.lactis NZ9000-OXM are significantly reduced, about of that in the L.lactis NZ9000 group. NZ3900-OXM decreases the food intakes of obese mice by 42.1% compared with the control group. Compared with the OXM transgenic Bifidobacterium reported in Chinese patent 200910041386.X, the OXM transgenic Bifidobacterium can decreases the food intakes of mice by 28.2% compared with the control group (FIG. 6). Thus, L.lactis NZ9000-OXM can better control the body weight by suppressing appetite and reducing the food intakes.

(41) (4) Six weeks after feeding of L.lactis, the distribution of the abdominal fat is measured with CT scanning. FIG. 7 is a cross-sectional scan of the abdominal cavity, and the light gray area delineated by white lines represents the adipose tissue. FIG. A represents the mice in the control group with normal diets. FIG. B represents the mice in the L.lactis NZ9000 feeding group, FIG. C represents the mice in the L.lactis NZ9000-OXM feeding group. It can be seen that the body fat content of mice in the NZ9000-OXM group is decreased significantly. The percentage of fats among the total body is calculated by CT analysis software and then fat weight for each mouse can be calculated. Data analysis is shown in FIG. 8. These results show that, the mice fed with L.lactis NZ9000 still have a large amount of abdominal fats, while the mice fed with L.lactis OXM have significantly less abdominal fat, and reduced to levels of normal mice, suggesting that L.lactis NZ9000-OXM has the effect of reducing body fat.

(42) (5) After induction of obesity in mice with high fat diets, the fasting blood glucose increases to about 7 mmol/L (FIG. 9), which is significantly higher than that of normal mice. However, after 6 weeks of feeding L.lactis, the fasting blood glucose of mice in the L. lactis NZ9000 group is about 6.5 mmol/L. The fasting blood glucose level of mice in L. lactis OXM group returns to normal level of 5 mmol/L. Comparative experiments show that the OXM transgenic Bifidobacterium reported in Chinese patent 200910041386.X can not regulate fasting blood glucose in mice. Therefore, OXM recombinant L.lactis is superior to OXM recombinant bifidobacteria in reducing the fasting blood glucose.

(43) (6) After mice are induced obesity with high fat diets, their serum cholesterol level is 2.42 mol/l. At the end of the experiment, mice are sacrificed and serum cholesterol levels were measured. As shown in FIG. 10, the serum cholesterol level of normal mice is 2.167 mol/l, the serum cholesterol level of mice fed with L. lactis NZ9000 is 3.478 mol/l, and the serum cholesterol level of mice fed with L. lactis NZ9000-OXM is decreased to 1.83 mol/l, significantly lower than that of mice fed with L. lactis NZ9000. The OXM recombinant Bifidobacteria reported in the Chinese patent 200910041386.X can reduce the cholesterol level to approximately 2.4 mol/l in equal time. Thus, the L. lactis NZ9000-OXM provided in the invention can significantly reduce the serum cholesterol level.

Example 3: Pharmacodynamics Experiments of OXM-Transformed Lactic Acid Bacteria to Weight Loss and Lipid Reduction in Gene-Deficient Diabetic Mice

(44) 1. Prepare twenty 8-week db/db mice, divide them into two groups, 10 mice in each group. One group is the control group, fed with L.lactis NZ9000; another group is the experiment group, fed with L.lactis NZ9000-OXM. At the same time, the normal control group is B6 mice fed normal diets without feeding with L.lactis.

(45) 2. Each mouse is received 510.sup.9 CFU L.lactis by intragastric administration every day. The body weight monitoring data are shown in FIG. 11. The body weights of mice fed with OXM can maintain unchanged compared with their own weight before administration. The body weights of mice in the NZ9000 group gradually increase, at the 5th week, increased by about 8%. The experiment shows that L.lactis NZ9000-OXM can effectively control the weight gain of diabetic mice.

(46) 3. At the same time, blood is drawn to detect the serum cholesterol levels (FIG. 12). The serum cholesterol levels in mice fed with OXM decrease significantly to 5.58 mol/l, significantly lower than that in the control group. This result suggests that L. lactis NZ9000-OXM can significantly reduce blood lipids in diabetic mice.

(47) The present invention has shown that, OXM transformed lactic acid bacteria can express and secrete OXM in the intestinal tracts of mice, and the expressed OXM can suppresses appetite and inhibits energy absorption by acting on the corresponding receptors in the body, suggesting that OXM recombinant L.lactis has significant biological effects of weight loss, lipid reduction, and glucose reduction.