CN114606148A

CN114606148A - Pichia pastoris strain for expressing osteopontin

Info

Publication number: CN114606148A
Application number: CN202210322609.5A
Authority: CN
Inventors: 刘龙; 陈坚; 吕雪芹; 堵国成; 李江华; 刘延峰; 张智航; 陶孟瑞; 孙丹
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-03-29
Filing date: 2022-03-29
Publication date: 2022-06-10

Abstract

The invention discloses a pichia pastoris strain for expressing osteopontin, belonging to the field of genetic engineering. The invention successfully integrates the gene containing P at the AOX1 site on the pichia pastoris genome_AOX1A promoter, an alpha signal peptide and an osteopontin expression frame of osteopontin with an amino acid sequence shown as SEQ ID NO. 1. Fermentation experiments show that the pichia pastoris gene engineering strain provided by the invention can successfully express osteopontin and partially secrete the osteopontin to the outside of cells, but protein secretion is delayed in the later period of fermentation, and most of protein enters vacuole. Through Western Blot verification and ELISA quantitative determination, the successful expression of the osteopontin is realized, and the expression quantity of the osteopontin reaches 32.2 mg/L.

Description

Pichia pastoris strain for expressing osteopontin

Technical Field

The invention relates to a pichia pastoris strain for expressing osteopontin, belonging to the field of genetic engineering.

Background

Osteopontin (OPN), also known as sialoglycoprotein, is an acidic protein that is highly phosphorylated and has O-glycosylation modifications, which is synthesized or secreted by various tissue cells in vivo and was first found in osteoblasts, and thus OPN was originally considered an important bone matrix protein and was closely associated with bone formation and development. In recent years, the content of OPN in breast milk, especially colostrum, is found to be very high, and the OPN is a very important type of immune active protein in the breast milk. There are significant differences in the OPN content of breast milk among different species, for example, the OPN content in cow's milk is about 18mg/L, whereas the concentration of OPN in human milk is as high as 138 mg/L. Meanwhile, the breast milk OPN also has obvious regional difference, the OPN level in the breast milk of China is obviously higher than that of other countries and regions, and the content of the OPN is also obviously different in different development periods of life. Research indicates that the OPN content in umbilical cord blood plasma of a newborn and plasma of a 3-month-old infant is very high and is about 7-10 times that of an adult, which indicates that the OPN has a positive effect on the early development of the infant, particularly on the aspects of intestinal growth, nervous system development, immunoregulation and the like in the early life.

Both intracellular OPN and secreted OPN play important roles in immune regulation, and there have been a number of animal studies and randomized clinical trials investigating the effects of breast milk OPN on immune development early in life. For example, injection of OPN into rat dermis induces the accumulation of macrophages that highly express OPN receptors towards the site of injection, and this local infiltration is significantly inhibited by anti-OPN neutralizing antibodies. Meanwhile, OPN can also induce dendritic cells to express human leukocyte DR antigen, so that the stimulation capability of the dendritic cells is enhanced, and the reflux of the dendritic cells from the epidermis to lymph nodes can be regulated and controlled through the interaction with CD44 and an integrin receptor. In addition, randomized clinical trials in infants (1-6 months) have also shown that OPN has a better modulating effect on infant immunity. The breast-fed, regular formula or OPN-supplemented formula contained 65 or 130mg/L OPN (50% or 100% of the average OPN concentration in breast milk, respectively). Compared with the infants fed with the conventional formula, the serum TNF-alpha level of the infants fed with the formula added with the OPN is remarkably reduced, and the sick days are reduced. The immune cell profile of infants fed with OPN-supplemented formula was more similar to breast-fed infants compared to infants fed with conventional formula. The OPN levels in plasma samples of breast-fed infants and infants with OPN formula added were higher than those of infants with regular formula at 4 and 6 months of age. These findings indicate that the addition of bovine milk OPN to infant formula may exert its beneficial effects by increasing endogenous OPN synthesis.

Currently, the market for obtaining OPN mainly comes from the extraction of fresh cow milk, and the separation of OPN from cow whey by anion exchange technology is performed, and the final obtained product contains about 78% of proteins, and nearly 95% of these proteins are OPN. The OPN obtained by this process contains ash, moisture and less than 1% fat and lactose.

The OPN obtained by the method is the only OPN finished product sold on the market at present, and the finished product has high phosphorylation and glycosylation through mass spectrum and functional research and analysis. However, the content of OPN in cow's milk is very low, about 11mg of OPN can be extracted from 1L of raw milk, and 1 kg of OPN can be extracted from about 90 tons of cow's milk according to the extraction amount. Therefore, large-scale marketing of OPN by fresh cow milk extraction is difficult and costly.

Disclosure of Invention

The invention aims to solve the technical problems that osteopontin is successfully expressed in pichia pastoris and correctly modified and processed, and meanwhile, the osteopontin can be quickly secreted out of cells to realize the high-efficiency expression of the enhanced osteopontin.

The first purpose of the invention is to provide a pichia pastoris gene engineering bacterium, wherein an expression cassette is integrated on the genome of the pichia pastoris gene engineering bacterium, and the expression cassette comprises P_AOX1Promoter, alpha signal peptide and osteopontin.

In one embodiment of the present invention, said P_AOX1The nucleotide sequence of the promoter is shown as SEQ ID NO.1, the amino acid sequence of the alpha signal peptide is shown as SEQ ID NO.2, and the amino acid sequence of the osteopontin is shown as SEQ ID NO. 3.

In one embodiment of the invention, the osteopontin is represented by P_AOX1The promoter promotes expression.

In one embodiment of the invention, the osteopontin is linked at its N-terminus to an alpha signal peptide.

In one embodiment of the invention, the pichia pastoris is host pichia pastoris X33.

In one embodiment of the invention, the pichia pastoris genetically engineered bacteria take a PICZ α a plasmid as an expression vector.

In one embodiment of the invention, the expression cassette is integrated at the AOX1 site of the pichia pastoris genome.

The second purpose of the invention is to provide a method for preparing osteopontin, which is to inoculate the pichia pastoris gene engineering bacteria in a culture medium for inducing expression of osteopontin.

In an embodiment of the invention, the method comprises the steps of firstly inoculating the pichia pastoris genetically engineered bacteria into a fermentation medium for fermentation culture to obtain a fermentation broth, and then inoculating the fermentation broth into an induction medium for induction expression.

In one embodiment of the invention, the OD of the fermentation broth₆₀₀Is 80 to 100.

In one embodiment of the invention, the fermentation medium is peptone 18-22 g/L, yeast powder 8-12 g/L, glycerol 8-12 mL/L, YNB 10-15 g/L, biotin4×10^-4g/L, and 80-120 mM/L potassium phosphate buffer solution.

In one embodiment of the invention, the induction medium is peptone 18-22 g/L, yeast powder 8-12 g/L, methanol 4-7 mL/L, YNB 10-15 g/L, biotin 4 × 10^-4g/L, and 80-120 mM/L potassium phosphate buffer solution.

In one embodiment of the present invention, the final concentration of methanol in the induction medium is 0.8-1.2%.

In one embodiment of the present invention, the fermentation culture conditions are 28-32 ℃ and 200-240 rpm.

In one embodiment of the present invention, the expression inducing condition is 28-32 deg.C, 200-240 rpm.

The invention also provides the pichia pastoris gene engineering bacteria and the application of the method in preparing products containing osteopontin.

Has the advantages that:

the present invention integrates osteopontin expression frame successfully at AOX1 site on Pichia pastoris genome. Fermentation experiments show that the osteopontin can be successfully expressed and partially secreted to the extracellular space, but protein secretion is delayed in the later period of fermentation, and most of the protein enters vacuoles. The successful expression of osteopontin is determined by Western Blot verification and ELISA quantitative determination, and the expression amount reaches 32.2 mg/L.

Drawings

FIG. 1 is an exemplary diagram of pPICZ. alpha.A-. alpha.factor-OPN vector.

FIG. 2 shows the verification of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with the sample ratios of 1-10, 11, M and protein Marker.

FIG. 3 shows a Western Blot (Western Blot) verification pattern, wherein 1-10 includes experimental group fermentation liquid supernatant, 11 includes blank control fermentation liquid supernatant, and M includes protein Marker.

FIG. 4 osteopontin standard curve.

FIG. 5 shows the result of ELISA detection of samples.

Detailed Description

The media referred to in the examples below:

BMGY medium: peptone 20g/L, yeast powder 10g/L, glycerol 10mL/L, YNB 13.4g/L, biotin 4X 10^-4g/L, potassium phosphate buffer 100 mM/L.

BMMY culture medium is: peptone 20g/L, yeast powder 10g/L, methanol 5mL/L, YNB 13.4g/L, biotin 4X 10^-4g/L, potassium phosphate buffer 100 mM/L.

EXAMPLE 1 construction of expression plasmid

(1) The osteopontin is obtained by using the artificially synthesized OPN gene as a template and the primers OPN-F and OPN-R in the table 1 for amplification, and the alpha-signal peptide is obtained by using the primers aOPN-Z-F and aOPN-Z-R in the table 1 for amplification by using the plasmid pPICZ alpha A preserved in a laboratory as a template. And connecting the osteopontin and the alpha-signal peptide obtained by amplification to the pPICZ alpha A plasmid subjected to double enzyme digestion by EcoRI and Not I to obtain a recombinant plasmid pPICZ alpha A-alpha factor-OPN. The plasmid map is shown in FIG. 1.

The PCR system is as follows:

TABLE 1 PCR reaction System

The PCR conditions were:

TABLE 2 PCR amplification conditions

The pPICZ alpha A-alpha factor-OPN recombinant plasmid is designed, and the plasmid map and the primer sequence are respectively shown in figure 1 and table 3.

TABLE 3 pPICZ. alpha.A-. alpha.factor-OPN primer sequences

Example 2 construction of recombinant Pichia pastoris

The recombinant plasmid pPICZ alpha A-alpha factor-OPN of example 1 was linearized with Dra I, 80. mu.L of Pichia pastoris X33 competent cells were mixed with 10. mu.L of the linearized recombinant plasmid homogeneously and transferred to an electrotransformation cup of 0.2cm ice-bathed at-20 ℃ and the cup containing the mixture was ice-bathed for 5 min. The parameters of the electric converter are adjusted and placed in the Pichia pastoris range, the voltage is 1.5 kilovolts, the capacitance is 25 microfarads, the resistance is 200 ohms, and the time is about 5 milliseconds. After electric shock, 1mL of 1M sorbitol solution precooled on ice was quickly added to the conversion cup, gently pipetted and mixed, and the mixture was aspirated and transferred to a centrifuge tube. Standing at 30 deg.C for 1-2h, and centrifuging at 2000g for 5 min. Discarding 800 μ L of supernatant, uniformly blowing the residual thallus, coating on YPD plate, and culturing in 30 deg.C incubator for 2-4 days until single colony grows out.

Example 3 fermentation production of osteopontin in recombinant Pichia pastoris

Single colonies were picked from YPD plates of example 2 and inoculated into round bottom tubes containing 2mL YPD medium overnight, 1mL overnight inoculum was inoculated into 25mL BMGY medium for shake flask fermentation at 30 ℃ and 220rpm to OD₆₀₀Centrifuging at 4 deg.C for 10min at about 90 deg.C for 3500g, collecting cells, discarding supernatant, washing with sterile water twice, centrifuging under the same conditions after each washing, and collecting supernatant. Cells were resuspended in BMMY medium and induction-expressed at 30 ℃ and 220rpm with methanol every 24h to a final concentration of 1%. After induction with methanol for 7d, the supernatant was centrifuged and collected, concentrated by centrifugation, and osteopontin was quantitatively analyzed by SDS-PAGE and Western Blot. As a result, as shown in FIGS. 2 and 3, a target band of OPN appeared at 35kDa, and the maximum yield reached 32.2 mg/L.

Using human osteopontin ELISA kit, the osteopontin standard was diluted in gradient until the concentrations were 0, 50, 100, 200, 400, 800 μ g/L, respectively, and detected together with the sample, finally obtaining the absorbance as in table 4, and making the regression curve as in fig. 4.

TABLE 4 absorbance of standard samples

Concentration of Standard substance (μ g/L)	0	50	100	200	400	800
							Absorbance (OD)₄₅₀)	0.065	0.265	0.536	0.898	1.413	2.126

After taking 1mL of each of 8 fermentation samples and 2 negative controls and diluting the samples by 100 times in a gradient, 10 samples were tested in parallel, and the results are shown in Table 5 and FIG. 5.

TABLE 5 fermentation sample detection Absorbance

Samples 1-8 are fermentation samples, 9-10 negative controls.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Claims

1. A pichia pastoris gene engineering bacterium, which is characterized in that an expression cassette is integrated on a pichia pastoris gene engineering bacterium genome, and the expression cassette contains P_AOX1Promoter, alpha signal peptide and osteopontin.

2. The pichia pastoris genetically engineered bacterium according to claim 1, wherein P is_AOX1The nucleotide sequence of the promoter is shown as SEQ ID NO.1, the amino acid sequence of the alpha signal peptide is shown as SEQ ID NO.2, and the amino acid sequence of the osteopontin is shown as SEQ ID NO. 3.

3. The pichia pastoris genetically engineered bacterium according to claim 1, wherein the osteopontin is composed of P_AOX1The promoter starts expression, and the N end of the osteopontin is connected with an alpha signal peptide.

4. The pichia pastoris genetically engineered bacterium according to any one of claims 1 to 3, wherein pichia pastoris X33 is used as a host, and pichia pastoris is used as an expression vector by using a PICZ alpha A plasmid.

5. The pichia pastoris genetically engineered bacterium of claim 1, wherein the expression cassette is integrated at the AOX1 site of the pichia pastoris genome.

6. A method for preparing osteopontin, which is characterized in that the method comprises the step of inoculating the pichia pastoris gene engineering bacteria of any one of claims 1 to 5 into a culture medium to induce and express osteopontin.

7. The method as claimed in claim 6, wherein the method comprises the steps of inoculating the pichia pastoris engineered bacteria as claimed in any one of claims 1 to 5 into a fermentation medium for fermentation culture to obtain a fermentation broth, and then inoculating the fermentation broth into an induction medium for induction expression.

8. The method of claim 6, wherein the OD of the fermentation broth₆₀₀Is 80 to 100.

9. The method of claim 7, wherein the final concentration of methanol in the induction medium is 0.8-1.2%.

10. The application of the pichia pastoris gene engineering bacteria as described in any one of claims 1 to 5 and the method as described in any one of claims 6 to 9 in preparing products containing osteopontin.