Application of alpha-amylase protein derived from nephroplasmosis in culture of nephroplasmosis

Abstract

The invention belongs to the technical field of biology, and provides an application of alpha-amylase protein derived from nephroplasmosis in culture of nephroplasmosis, and the alpha-amylase protein is obtained by synthesizing a gene sequence of alpha-amylase as shown in SEQ ID NO.2 after codon optimization and then transfecting the gene sequence to an expression vector. And carrying out bacterial transformation, culture, induction and crushing. The invention also provides a method for inducing the formation of the reproductive cyst of the reniform worm. It is found for the first time that alpha-amylase protein specifically induces reproductive cysts to be formed, the biomass of the nephroplasmosis is increased, the growth generation of the nephroplasmosis is shortened, and the method is suitable for species such as distended nephroplasmosis and nephroplasmosis henslouianum and fits natural ecological scenes; complex equipment is not needed, the cost is low, large-scale production is easy, the stability is high, and the method can be widely applied to soil ecological restoration, agricultural microbial resource development and basic research related to nephroplasmosis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and provides an application of α-amylase protein derived from kidney-shaped insects in kidney-shaped insect culture, which can be applied to soil ecological restoration, agricultural microbial resource development and basic research related to kidney-shaped insects. Background Technology

[0002] Reniform parasite ( Colpoda Nephropoda is a single-celled eukaryotic organism belonging to the phylum Ciliophora, class Nephridia, order Nephridia, family Nephropodidae, and genus Nephropoda. It is named for its kidney-shaped body and the regular distribution of cilia on its body surface. Nephropoda are widely distributed and occupy an absolutely dominant ecological niche abundance in global soil ecosystems.

[0003] As an important functional group in soil ecosystems, kidney-shaped organisms (Nephrodisiacs) are characterized by short life cycles, rapid community succession, wide distribution, and strong environmental responsiveness. Their life cycle mainly consists of two types: dormant cysts used to cope with adversity and reproductive cysts related to reproduction. The latter form multiple daughter cells through cell division, a crucial strategy for population expansion and environmental adaptation. These characteristics endow them with multiple ecological functions: regulating soil microbial community structure through feeding on microorganisms, accelerating the decomposition of organic matter and nutrient transformation, and providing plants with usable nitrogen, phosphorus, and other nutrients; exhibiting high tolerance to heavy metals and organic pollutants, and reducing soil toxicity through accumulation and transformation, making them a superior group for soil bioremediation; and serving as environmental indicator organisms, reflecting the degree of soil pollution and ecological health, providing a scientific basis for environmental management. Therefore, kidney-shaped organisms hold an irreplaceable position in basic research on soil ecology and microbiology.

[0004] Despite the significant ecological value and promising applications of *Nephroplasma gondii*, current cultivation techniques still suffer from several shortcomings: Firstly, obtaining high biomass is difficult; conventional culture methods struggle to achieve high-density proliferation. For instance, patent application number 202111551987.2, entitled "A Method for Long-Term Preservation and Rapid Recovery of *Nephroplasma gondii* Strains," mentions that under wheat grain culture medium, *Nephroplasma gondii* (…)… Colpoda inflata The maximum population density of *Nephrodisiac* cells is typically around 4000 cells / mL, requiring 72 hours for incubation. Patent application number 202411858916.0, entitled "A Simple Method for Expanding the Culture of *Nephrodisiac* and Resuscitating *Nephrodisiac* Cysts," mentions that using wheat seedling culture medium combined with *E. coli* to prepare a *Nephrodisiac* culture medium can shorten the culture time and significantly increase the maximum density of *Nephrodisiac* cells, but the maximum population density only reaches approximately 20,000 cells / mL. Secondly, the key molecular mechanisms of reproductive cyst formation are not yet clear, making targeted and efficient induction and regulation impossible. These problems limit the application of *Nephrodisiac* in soil remediation, agricultural disease control, and other scenarios.

[0005] Previous studies, through genomic, transcriptomic, and proteomic data analysis, have revealed that the secreted protein α-amylase in *Nephrodisiac* may promote the formation of genital cysts. This invention establishes a method for rapidly increasing the formation rate and biomass of *Nephrodisiac* genital cysts through the in vitro preparation of α-amylase protein. This not only shortens the culture cycle and reduces operating costs, providing technical support for the modeling research of *Nephrodisiac*, but also promotes its practical application in soil health monitoring, heavy metal bioremediation, and environmental indicator organisms. It has significant scientific value and practical implications for the development of novel bioremediation technologies and environmental monitoring tools. Summary of the Invention

[0006] To address the aforementioned technical problems, the first objective of this invention is to provide an application of α-amylase protein derived from *Nephrodisiac* in *Nephrodisiac* culture. The α-amylase protein is prepared by synthesizing the gene sequence of α-amylase as shown in SEQ ID NO.2 with optimized codons, transfecting it into an expression vector, and then performing bacterial transformation, culture, induction, and disruption.

[0007] The amino acid sequence of α-amylase protein derived from Nephroticosa (as shown in SEQ ID NO.1) was optimized using bacterial codons to obtain the optimized α-amylase gene sequence shown in SEQ ID NO.2.

[0008] The initial density of *Nephrodisiac* culture was 100-200 individuals / mL. *Nephrodisiac* culture included pre-culture and cell culture. Pre-culture was conducted in a pre-culture medium containing 0.1%-0.2% wheatgrass culture medium. Cell culture was conducted in an α-amylase protein stimulation medium at a concentration of 5× to 100×.

[0009] A second objective of this invention is to provide a method for inducing the formation of genital cysts in *Nephrodisiac*, comprising the following steps: (1) Prepare the optimized α-amylase protein described above; (2) Pre-culture of Nephroticosa: A certain amount of Nephroticosa was taken into the pre-culture medium and the initial density was 100-200 Nephroticosa / mL. The medium was kept at a constant temperature of 20-25℃ and cultured until the density reached 1000-2000 Nephroticosa / mL. The pre-culture of Nephroticosa was completed and the Nephroticosa culture medium was obtained at the same time. (3) Preparation of α-amylase protein stimulation solution: Filter the worm body into the culture medium of the kidney worm obtained in step (2) to obtain the filtrate. Add the filtrate to the α-amylase protein obtained in step (1) and mix well to obtain the α-amylase protein stimulation solution. Prepare and use immediately. (4) Nephroticella cell culture: Take the new Nephroticella culture medium obtained in step (2) and add it to the α-amylase protein stimulation solution obtained in step (3). Incubate in a constant temperature incubator at 20-25℃ for 16-36 hours to reach the maximum cell growth density.

[0010] In step (1), the preparation process is as follows: the α-amylase protein sequence is codon-optimized and synthesized, then transfected into the PET32a vector. The plasmid is then transformed into bacteria BL21 using the heat shock method. Single clones are picked and inoculated into 3 mL of LB medium containing Amp (100 μg / mL) and cultured overnight at 37°C and 200 rpm. The next day, 1 mL of bacterial culture is transferred to 50 mL of LB medium containing Amp (100 μg / mL) and cultured at 37°C and 200 rpm for 3 h. The OD600 of the cultured bacterial culture is measured to be 0.6-0.8 using a spectrophotometer (generally considered to be at the logarithmic growth phase). IPTG was added to the cultured bacterial solution to a final concentration of 0.5 mM, and the mixture was induced at 28°C and 200 rpm for 4 h with shaking. The induced bacterial solution was then centrifuged at 4000 rpm for 10 min, and the precipitate was collected and transferred to a centrifuge tube. The precipitate was then sonicated under ice bath conditions. The bacterial cells were resuspended in 2 mL of PBS in each tube, and sonicated at 24% power (2 s on / 2 s off, 10 min, 4°C). After centrifugation at 15000 rpm and 4°C for 10 min, one tube of α-amylase protein precipitate was obtained. The precipitate was stored at -20°C for later use.

[0011] In step (2), the pre-culture solution is a 0.1%-0.2% wheat seedling culture solution. The preparation method is as follows: weigh 10g of wheat seedlings into 200mL of ddH2O and boil for 10min; centrifuge the boiled wheat seedling stock solution at 3000rpm for 5min, retain the supernatant, filter it through a 0.22μm filter membrane, add ddH2O to make up to 1L, and obtain a 1% wheat seedling culture solution. After high-temperature steam sterilization, store at room temperature. To prepare a 0.1%-0.2% wheat seedling culture solution, add 10-20 mL of 1% wheat seedling culture solution to 90-80 mL of sterile ddH2O until the volume is 100 mL.

[0012] Preferably, step (2) is as follows: inoculate the kidney-shaped parasites into 10 mL of 0.1%-0.2% wheat seedling culture medium to make the initial amount 100-200 parasites / mL, place it in a constant temperature incubator at 25℃ and let it stand for culture. When the number of parasites reaches 1000-2000 parasites / mL, it is judged to be in the early logarithmic phase. At this time, the kidney-shaped parasite culture medium required for subsequent use is obtained.

[0013] Preferably, step (3) is as follows: the *Nephroticosa* culture medium obtained in step (2) is filtered through a 0.45 μm sterile needle filter to remove the worms, and the supernatant is retained. 2 mL of the filtrate is added to one tube of α-amylase protein precipitate obtained in step (1), and the mixture is homogeneous. The resulting solution is defined as a 10× concentration α-amylase protein stimulating solution. This stimulating solution must be prepared and used immediately to avoid a decrease in protein activity due to storage. When using, the concentration range of the α-amylase protein stimulating solution can be adjusted to 5× concentration - 100× concentration by dilution or concentration as needed.

[0014] Among them, step (4) is as follows: take 3 mL of the new kidney-shaped worm culture medium obtained by the same operation in step (2), add 600 μL of the α-amylase protein stimulation solution obtained in step (3) and mix well, and incubate in a constant temperature incubator at 25℃. The maximum cell growth density can be reached after 16h-36h of culture. Every 3h, the cell density is counted once with a floatation counting plate, the growth curve is plotted and the reproductive cyst rate is statistically analyzed.

[0015] The advantages and beneficial effects of this invention compared to the prior art include: 1. High efficiency: This method can increase the formation rate of Nephroticosa genital cysts to up to 39%, and the maximum cell growth density can exceed 6*102. 4 The concentration of *Nephrodisiac* per mL significantly increased the biomass of *Nephrodisiac* and shortened its generation time. 2. High specificity: α-amylase protein can specifically induce the formation of reproductive cysts, thereby increasing the biomass of Nephroticosa. 3. Good versatility: It can be applied to a variety of species of the Nephroticosa genus (such as Nephroticosa dilatatosa, Nephroticosa henri, etc.), which fits the natural ecological scene; 4. Simple operation: No complicated equipment is required. Induction can be achieved simply by adding a large amount of induced α-amylase protein. It is low in cost, easy to scale up, and suitable for laboratory research and field application. Attached Figure Description

[0016] To more clearly describe the embodiments in this specification or the technical solutions in the prior art, the accompanying drawings used are briefly introduced below. The drawings in the following description represent only some embodiments of the present invention. For those skilled in the art, other related drawings can still be obtained from these drawings without creative effort.

[0017] Figure 1 The flowchart for the preparation and induced culture of α-amylase protein in *Nephrodisiac* provided by this invention; Figure 2 In Embodiment 1 of the present invention C. inflata YX4-derived α-amylase protein stimulation solution C. inflatedGrowth curve and reproductive cyst rate statistics of YX4; Figure 3 In Embodiment 2 of the present invention C. inflata Stimulation with α-amylase protein at different concentration gradients derived from YX4 C. inflata Growth curve and reproductive cyst rate statistics of YX4; Figure 4 In Embodiment 3 of the present invention C. inflata Growth curves of four types of kidney parasites stimulated by α-amylase protein stimulating solution derived from YX4. Figure 5 In Embodiment 3 of the present invention C. inflata Statistical chart of reproductive cyst formation rates of four types of kidney-shaped parasites stimulated by α-amylase protein stimulating solution derived from YX4. Figure 6 Stimulation with α-amylase protein stimulation solutions from four sources as described in Example 4 of this invention. C. inflata Growth curve and reproductive cyst rate statistics of YX4. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but the present invention does not limit the implementation thereto.

[0019] The material source information in the embodiment is as follows: pET32a expression vector: derived from Sangon Biotech (Shanghai) Co., Ltd. BL21 bacteria: sourced from Beijing Qingke Biotechnology Co., Ltd. C. inflata YX4: Sourced from the National Aquatic Organism Germplasm Bank Colpoda henneguyi YX6: Sourced from the National Aquatic Organism Germplasm Bank Colpoda steinii HBM110: Sourced from the National Aquatic Organism Germplasm Bank Colpoda maupasi TBM90: Sourced from the National Aquatic Organism Germplasm Bank Example

[0020] In this embodiment, treatment was performed using a 10× α-amylase protein stimulation solution. C. inflata YX4, each experiment was set up with the same 3 replicates. The specific steps included: 1) Preparation of culture medium, buffer solution, and reagents: The procedure for preparing a 0.1% wheatgrass culture medium is as follows: Weigh 10g of wheatgrass (Lifestream Organic Wheat Grass Powder 250g, purchased from Amazon.com https: / / www.amazon.co.uk / Lifestream-Organic-Wheat-Grass-Powder / dp / B000NRXMOQ) into 200mL of ddH2O and boil for 10 minutes. Aliquot the boiled wheatgrass stock solution into 50mL centrifuge tubes, centrifuge at 3000rpm for 5 minutes, retain the supernatant, filter through a 0.22μm filter membrane, and add ddH2O to the filtered wheatgrass stock solution to bring the volume to 1L, obtaining a 1% wheatgrass culture medium. After high-temperature steam sterilization, store at room temperature. When using, it can be diluted to different concentrations. To prepare a 0.1% wheatgrass culture medium, add the 10mL of the 1% wheatgrass culture medium to 90mL of sterile ddH2O to a volume of 100mL, and store at room temperature for later use. Preparation of LB medium: Weigh 10g tryptone, 5g yeast extract, and 10g NaCl and dissolve them in 800mL ddH2O. Stir thoroughly to dissolve, then bring the volume up to 1L with ddH2O. Sterilize at high temperature and store at room temperature. The procedure for preparing a 100 mg / mL Amp solution is as follows: Weigh 1 g of ampicillin sodium salt powder, add it to 8 mL of ddH2O, stir thoroughly to dissolve, then bring the volume to 10 mL with ddH2O. Filter the solution through a 0.22 μm filter for sterilization, and dispense it into 1.5 mL sterile centrifuge tubes (1 mL per tube) to obtain a 100 mg / mL Amp solution. Store at -20℃. When using, the working concentration of Amp is 100 μg / mL. Preparation of 0.5M IPTG solution: Weigh 2.383g of IPTG powder, add it to 8mL of ddH2O, stir thoroughly to dissolve, then bring the volume up to 10mL with ddH2O, filter sterilize using a 0.22μm filter, dispense into 1.5mL sterile centrifuge tubes (1mL per tube), and store at -20℃. The procedure for preparing 1× PBS buffer is as follows: First, prepare 20× PBS buffer by weighing 8g NaCl, 0.2g KCl, 3.58g Na₂HPO₄·12H₂O, and 0.27g KH₂PO₄ and dissolving them in 800mL ddH₂O. Stir thoroughly to dissolve, then bring the volume to 1L with ddH₂O. Sterilize at high temperature and store at room temperature to obtain 20× PBS buffer. 1× PBS buffer is obtained by adding 1mL of 20× PBS buffer to 19mL of sterile ddH₂O.

[0021] 2) Preparation of α-amylase protein: α-amylase protein (derived from *Nephrodisiac*) was prepared. C. inflata The YX4 sequence (SEQ ID NO.1) was optimized using bacterial codons. The optimized gene sequence information is shown in SEQ ID NO.2 (where EcoRI / SalI are restriction enzyme sites). Gene synthesis was performed and transfected into the pET32a expression vector. The pET32a vector containing the α-amylase gene (SEQ ID NO.2) was transformed into BL21 bacteria using a heat shock method, and the mixture was plated. After picking a single colony, it was inoculated into 3 mL of LB medium containing 100 μg / mL Amp and cultured overnight at 37°C and 200 rpm with shaking. The next day, 1 mL of the bacterial culture was transferred to 50 mL of LB medium containing 100 μg / mL Amp and cultured at 37°C and 200 rpm for 3 hours until the OD600 reached 0.6-0.8, at which point the colony count was 10^6. 6 CFU / mL or higher. Add 0.5M IPTG solution to the bacterial culture until the final IPTG concentration is 0.5mM, and shake at 28℃ and 200rpm for 4 hours. Pour the bacterial culture into a 50mL centrifuge tube, centrifuge at 4000rpm for 10 minutes, collect the bacterial pellet, and resuspend the bacteria in 2mL of 1×PBS buffer. Sonicate the pellet at 24% power with 2s on / 2s off parameters for 10 minutes (maintaining 4℃ throughout). After sonication, centrifuge the bacterial culture at 15000rpm at 4℃ for 10 minutes, collecting the supernatant and pellet separately; this yields one tube of α-amylase protein pellet. Perform Coomassie brilliant blue staining and Western blotting experiments on small amounts of the supernatant and pellet, revealing the presence of α-amylase protein in the bacterial pellet. Therefore, store the pellet at -20℃ for later use.

[0022] 3) Pre-culture of kidney-shaped parasites: Take a small amount of... C. inflata YX4 worms were placed in a sterile petri dish containing 5 mL of 0.1% wheatgrass culture medium obtained in step 1). Once the density reached a certain level, they were transferred to a 50 mL Erlenmeyer flask containing 0.1% wheatgrass culture medium, resulting in an initial inoculation of 100-200 worms / mL. The worms were then incubated at 25°C. The density of the kidney-shaped worms was counted every 6 hours until the density reached 1000-2000 worms / mL. At this point, the culture medium was the kidney-shaped worm culture medium required for subsequent steps.

[0023] 4) Preparation of α-amylase protein stimulation solution: Pass the Nephroticosa culture medium obtained in step 3) through a 0.45μm sterile needle filter, retain the supernatant filtrate, add 2mL of filtrate to each tube of α-amylase protein precipitate obtained in step 2), and mix well to prepare a 10× concentration α-amylase protein stimulation solution, which should be prepared and used immediately.

[0024] 5) Nephroticosa cell culture: Take 3 mL of the Nephroticosa culture medium obtained in step 3) into a 6-well cell culture plate, add 600 μL of the 10× concentration α-amylase protein stimulation solution obtained in step 4), and incubate at 25℃ in a constant temperature incubator. Perform cell density counting every 3 hours to plot the growth curve of this Nephroticosa cell culture, and calculate the reproductive cyst rate and generation time. Calculate the population growth rate r of the protozoan according to the following formula: r = (ln Nt - ln N0) / t (r refers to the population growth rate, t refers to time, N0 = 0 h, and Nt = t). Calculate the r value to obtain the natural growth equation of the population, and then calculate the generation time according to G = ln 2 / r (G refers to the generation time, and r refers to the population growth rate). The genital cyst formation rate is derived from s = Nr / Nt * 100% (where s refers to the genital cyst formation rate, Nr refers to the genital cyst density at time t, and Nt refers to the population density of *Nephroticosa* at the same time).

[0025] In this embodiment, 0.1% wheatgrass culture medium was used as the control group (3 mL of the *Neopterygium* culture medium obtained in the same procedure as in step 3 was placed in a 6-well cell culture plate, at which point the *Neopterygium* density was 1000-2000 individuals / mL, and allowed to continue growing, serving as the control group). 10× α-amylase protein stimulation solution was used as the experimental group. The growth curve of *Neopterygium* and the statistical analysis of the reproductive cyst formation rate are shown below. Figure 2 As shown in the figure, the experimental group reached its peak density of 60,280 individuals / mL at 36 hours, while the control group reached its peak density of 13,200 individuals / mL at 25 hours. Calculating the generation time over the 16-25 hour period, the experimental group had a generation time of 4.97 hours, while the control group had a generation time of 9.1 hours. Although the logarithmic growth phase of the *Nephroplasma reniformis* population was delayed by approximately 11 hours in the experimental group, its peak population density was higher than the control, and its generation time was shorter. The experimental group reached its peak reproductive cyst rate of 39% at 22 hours, while the control group reached its peak of 16% at 16 hours, indicating that the experimental group significantly improved the cyst formation rate and prolonged the reproductive period.

[0026] Example 2 In this embodiment, α-amylase protein stimulation solutions of different concentration gradients were used for treatment. C. inflata YX4, each experimental group was set up with the same 3 replicates. Specifically, the following steps were included: except for steps 2) and 4), the other steps were the same as in Example 1; wherein, 2) Preparation of α-amylase protein and BL21 bacterial precipitate: Same as step 2) in Example 1 to obtain one tube of α-amylase protein precipitate. Additionally, BL21 bacteria were inoculated into 3 mL of LB medium and cultured overnight at 37°C and 200 rpm (as a negative control). The next day, 1 mL of BL21 bacterial culture was transferred to a new 50 mL LB medium and cultured at 37°C and 200 rpm for 3 hours until the OD600 reached 0.6-0.8, at which point the colony count was 10^6. 6CFU / mL or higher. Add 0.5M IPTG solution to the bacterial culture until the final IPTG concentration is 0.5mM, and shake at 28℃ and 200rpm for 4 hours. Pour the bacterial culture into a 50mL centrifuge tube, centrifuge at 4000rpm for 10 minutes, collect the bacterial pellet, and then add 2mL of 1×PBS buffer to the pellet to resuspend the bacteria. Sonicate the mixture at 24% power with 2s on / 2s off parameters for 10 minutes (maintaining 4℃ throughout). After sonication, centrifuge the bacterial culture at 15000rpm at 4℃ for 10 minutes, and collect the pellet; this yields one tube of BL21 bacterial pellet. Store the pellet at -20℃ for later use.

[0027] 4) Preparation of α-amylase protein stimulation solution: The *Nephrodisiac* culture medium obtained in step 3) was filtered through a 0.45 μm sterile syringe filter to obtain filtrate. A quantitative amount of the above filtrate was added to the α-amylase protein precipitate and BL21 bacterial precipitate obtained in step 2), and mixed thoroughly to prepare the α-amylase protein precipitation stimulation solution and the BL21 bacterial precipitate supernatant, which were prepared fresh for immediate use. Specifically, 2 mL of filtrate was added to one tube of α-amylase protein precipitate for mixing, defined as a 10× concentration stimulation solution. Simultaneously, stimulation solutions with different concentration gradients (0.25×, 0.5×, 1×, 5×, 10×, 20×, 50×, and 100×) were prepared for later use using dilution or concentration methods.

[0028] In this embodiment, 0.1% wheat seedling culture medium (control group 1, same as in Example 1) and BL21 bacterial precipitate supernatant (control group 2) were used as controls, and different concentrations of α-amylase protein stimulation solutions were used as experimental groups. The solutions from step 3) were taken respectively. C. inflated YX4 kidney-shaped parasites, grown to the early logarithmic phase (approximately 1000-2000 individuals / mL), were cultured in 3 mL of medium. Then, α-amylase protein stimulation solutions at concentration gradients of 0.25×, 0.5×, 1×, 5×, 10×, 20×, 50×, and 100× were added for treatment. The growth curves of the kidney-shaped parasites and the statistical analysis of the reproductive cyst formation rate are shown below. Figure 3 As shown in the figure. The results showed that the highest population density in the five experimental groups with α-amylase protein stimulation at concentrations ranging from 5× to 100× was higher than that in the two control groups. Furthermore, the maximum growth density of *Gnaphalium affine* gradually increased with increasing α-amylase protein concentration, with the highest density at 5× concentration reaching 68,433 individuals / mL at 16 hours. Regarding the effect on inducing genital cyst formation, the induction effect gradually increased with increasing concentration in all five experimental groups within the 5× to 100× concentration range; a 1× concentration produced a weak inhibitory effect; no genital cysts formed at a 0.5× concentration, cells became abnormally enlarged and transparent, and lysed within 48 hours; at a 0.25× concentration, cells were severely deformed and all died within 5 hours. These results indicate that α-amylase protein... C. inflataYX4 growth is dose-dependent: appropriate concentrations promote the formation of reproductive cysts, while too low or too high concentrations lead to cell malformation or even death.

[0029] Example 3 In this embodiment, four types of kidney-shaped worms were selected based on their phylogenetic relationships. C. inflated YX4, Colpoda henneguyi YX6 Colpoda steinii HBM110 Colpoda maupasi TBM90), a source prepared using the same method as step 4) in Example 1. C. inflata YX4 at a concentration of 10× α-amylase protein stimulation solution was used to treat four species of kidney-shaped worms in the early logarithmic phase. Each experiment had three replicates. The specific steps included: except for steps 3) and 5), the other steps were the same as in Example 1; wherein, 3) Pre-culture of kidney-shaped parasites: Take four types of kidney-shaped parasites into culture dishes containing 0.1% wheatgrass culture medium obtained in step 2). When the density reaches a certain level, transfer them to 50mL Erlenmeyer flasks containing 0.1% wheatgrass culture medium, so that the initial inoculation amount is 100-200 parasites / mL. Incubate in a constant temperature incubator at 25℃. Count the density of kidney-shaped parasites every 3 hours until the logarithmic growth phase, when the density is about 1000-2000 parasites / mL. At this time, the culture medium is the required culture medium.

[0030] 5) Nephroticosa cell culture: Take 3 mL of Nephroticosa culture medium obtained in step 3) into a 6-well cell culture plate, add 600 μL of 10× concentration α-amylase protein stimulation solution obtained in step 4) to different wells, and incubate at 25℃ in an incubator. Perform cell density counting every 3 hours to plot the growth curves of different Nephroticosa cell cultures and calculate the reproductive cyst rate.

[0031] In this embodiment, four species of kidney-shaped parasites were used as the control group with 0.1% wheatgrass culture medium (same as the control group in Example 1), and the experimental group was treated with 10× concentration α-amylase protein stimulation solution. The growth curves were plotted as follows. Figure 4 As shown, the fertilization cyst rate is as follows: Figure 5 As shown, the highest growth density of the four types of kidney-shaped parasites in the experimental groups was greater than that in the control group. C. inflata The highest growth density in group YX4 was 58,166 individuals / mL; C. henneguyi The highest growth density in group YX6 was 41,366 individuals / mL; C. stone The highest growth density in the HBM110 group was 40,366 individuals / mL; C. maupasiThe highest growth density in the TBM90 group was 19,733 individuals / mL. The highest genital cyst rates in all four kidney-shaped parasite experimental groups were also higher than those in the control group. C. inflata The maximum fertilized cyst formation rate in the YX4 group was 32%, which was 1.78 times that of the corresponding control group; C. henneguyi The maximum fertilized cyst formation rate in the YX6 group was 29%, which was 1.41 times that of the corresponding control group; C. steinii The maximum fertilized cyst rate in the HBM110 group was 24%, which was 1.50 times that of the corresponding control group; C. maupasi The maximum fertilized cyst rate in the TBM90 group was 35.6%, which was 1.48 times that of the corresponding control group. (Note: This likely refers to a specific characteristic or indicator.) C. inflata α-Amylase protein can induce the formation of reproductive cysts in different species of the genus *Nephrodisia*, but the induction effect varies. Based on a comparison of the highest growth densities of *Nephrodisia*, the order of induction strength is as follows: C. inflated YX4> C. henneguyi YX6> C. steinii HBM110> C. maupasi TBM90 suggests that the degree of response of the α-amylase protein in *Nephrodisiac* may be related to the phylogenetic relationship between species.

[0032] Example 4 In this embodiment, four types of kidney-shaped worms were selected based on their phylogenetic relationships. Colpoda sp. 21、 Colpoda bresslauides , C. magna , C. steinii α-amylase protein stimulating solutions derived from the above four species of *Nephroticosa* were prepared using the methods described in steps 2)-4) of Example 1, respectively, for stimulation. C. inflata YX4, each experiment was set up with the same 3 replicates. Specifically, the following steps were included: except for steps 2), 4), and 5), the other steps were the same as in Example 1: Wherein, 2) Preparation of α-amylase protein: Using a method similar to that in Example 1, the four types of kidney-shaped parasites were respectively prepared... C. The amino acid sequence of sp.21 α-amylase protein (SEQ ID NO.3) C. bresslauides The amino acid sequence of α-amylase protein (SEQ ID NO.5) C. magna The amino acid sequence of α-amylase protein (SEQ ID NO.7) C. steinii The amino acid sequence of the α-amylase protein (SEQ ID NO.9) was optimized by codon extraction to obtain the optimized sequence. C. The gene sequence of sp.21 α-amylase (SEQ ID NO.4), and the optimized sequence. C. bresslauides The gene sequence of α-amylase (SEQ ID NO.6), and the optimized... C. magna The gene sequence of α-amylase (SEQ ID NO.8), and the optimized... C. stone The gene sequence of α-amylase (SEQ ID NO. 10); all optimized gene sequences used EcoRI / SalI as restriction sites. The four optimized gene sequences were synthesized and transfected into the PET32a vector. The vector was then transformed into bacteria BL21 using a heat shock method. After plating, single colonies were picked and inoculated into 3 mL of LB medium containing Amp (100 μg / mL) and cultured overnight at 37°C and 200 rpm. The next day, 1 mL of the bacterial culture was transferred to 50 mL of LB medium containing Amp (100 μg / mL) and cultured at 37°C and 200 rpm for 3 h (shaking until OD600 was 0.6-0.8). 0.5 mM PTG solution was added to the bacterial culture, bringing the final concentration to 0.5 mM, and the culture was incubated at 28°C and 200 rpm for 4 h. The bacterial culture was poured into a 50 mL centrifuge tube and centrifuged at 4000 rpm for 10 min. The bacterial pellet was then collected and transferred to a 2 mL centrifuge tube. The pellet was sonicated on ice. The cells were resuspended in 2 mL of 1× PBS buffer and sonicated at 24% power with 2 s on / 2 s off parameters for 10 min (keeping the tube on ice throughout). After sonication, the pellet was centrifuged at 15000 rpm at 4°C for 10 min. The supernatant and pellet were collected separately. 10 μL of each sample was stained with Coomassie Brilliant Blue and subjected to Western blotting. The results showed that α-amylase protein was present in the bacterial pellet. Therefore, the pellet was stored at -20°C for later use.

[0033] 4) Preparation of α-amylase protein stimulation solution: 3 mL of the four different Nephroticosa culture media obtained in step 3) were passed through a 0.45 μm sterile needle filter, and the supernatant was retained. 2 mL of the filtrate was added to the four α-amylase protein precipitates obtained in step 2) and mixed evenly to prepare four 10× concentration α-amylase protein stimulation solutions. The solutions were prepared and used immediately.

[0034] 5) Nephroticosa cell culture: Take 3 mL of Nephroticosa culture medium obtained in step 3) into a 6-well cell culture plate, add 600 μL of the four 10× concentration α-amylase protein stimulation solutions obtained in step 4) to each well, and incubate statically in a 25℃ incubator. Perform cell density counting every 3 hours to plot the growth curve of this Nephroticosa cell culture and calculate the reproductive cyst rate.

[0035] In this embodiment, 10× concentrations of four α-amylase protein stimulating solutions were used as experimental groups, and 0.1% wheat seedling culture medium was used as the control group (the control group was set up in the same way as in Example 1). C. inflata YX4 growth curve and reproductive cyst rate plotted as follows: Figure 6As shown. The highest growth density in the experimental group was higher than that in the control group, among which... C. sp . 21 groups and C. bresslauides The highest density in all groups was 31,700 individuals / mL; C. magna The highest density in all groups was 18,600 individuals / mL; C. steinii The highest density in both experimental and control groups was 16,900 individuals / mL, while the highest density in the control group was 8,767 individuals / mL. The genital cyst rate in the experimental group was higher than that in the control group. C. sp . The cyst rate of the 21-unit reproductive tract reached a peak of 31.7% at 16 hours. C. bresslauides The group reached its peak at 28% at 8 hours; C. magna The group reached its peak at 24% at 8 hours; C. steinii The peak value of the group was 25% at 8 hours; the peak value of the control group was 16% at 10 hours. The results indicate that all four α-amylase proteins can induce [therapeutic effects] to varying degrees. C. inflata YX4 forms reproductive cysts and promotes cell proliferation. Based on a comparison of the highest growth densities of *Gnaphalium affine*, the order of induction strength of α-amylase proteins from different *Gnaphalium affine* species was as follows: C. sp . 21> C. bresslauides > C. magna > C. steinii It is closely related to phylogeny.

[0036] The above examples illustrate that, from *Nephroplasma gondii*... C. inflata The α-amylase protein obtained from YX4 can induce the formation of genital cysts and rapid population proliferation in *Nephrodisiac*, and this effect is dose-dependent, with the maximum induced cell density reaching 4-6 times that under standard culture conditions. Further studies revealed that the α-amylase protein can induce the formation of genital cysts in different *Nephrodisiac* species across species, and the induction efficiency is negatively correlated with the phylogenetic distance between species. Heterologous expression experiments of α-amylase protein in different *Nephrodisiac* species confirmed its cross-species induction ability.

[0037] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of an α-amylase protein derived from Nephrocytium sp. in the cultivation of Nephrocytium sp., characterized in that, The alpha-amylase protein is prepared by synthesizing the gene sequence of the alpha-amylase shown in SEQ ID NO. 2 after codon optimization, transfecting into an expression vector, bacterial transformation, culture, induction, and breaking.

2. Use according to claim 1, characterized in that, The initial density of the kidney-shaped worm culture is 100-200 worms / mL.

3. Use according to claim 1, characterized in that, The kidney-shaped worm culture includes kidney-shaped worm pre-culture and kidney-shaped worm cell culture, the kidney-shaped worm pre-culture is cultured in a pre-culture solution, the pre-culture solution includes 0.1%-0.2% wheat seed culture solution, and the kidney-shaped worm cell culture is cultured in an alpha-amylase protein stimulating solution.

4. Use according to claim 3, characterized in that, The preparation steps of the alpha-amylase protein stimulating solution are as follows: obtaining kidney-shaped worm culture solution after pre-culturing the kidney-shaped worm, filtering the kidney-shaped worm culture solution to obtain filtrate, adding the filtrate into the alpha-amylase protein and mixing uniformly to obtain the alpha-amylase protein stimulating solution.

5. A method of inducing the formation of Schistosoma reproductive packets, comprising the steps of, The method comprises the following steps: (1) preparing the alpha-amylase protein according to claim 1; (2) kidney-shaped worm pre-culture: sucking a certain amount of kidney-shaped worms into a pre-culture solution to make the initial density of the kidney-shaped worms be 100-200 worms / mL, and culturing at a constant temperature of 20-25°C, until the density of the kidney-shaped worms is 1000-2000 worms / mL, the kidney-shaped worm pre-culture is completed, and kidney-shaped worm culture solution is obtained at the same time; (3) preparing an alpha-amylase protein stimulating solution: filtering the kidney-shaped worm culture solution obtained in step (2) to obtain filtrate, adding the filtrate into the alpha-amylase protein obtained in step (1) and mixing uniformly to obtain the alpha-amylase protein stimulating solution, which is prepared and used immediately; (4) kidney-shaped worm cell culture: adding the new kidney-shaped worm culture solution obtained by the same operation in step (2) into the alpha-amylase protein stimulating solution obtained in step (3), and culturing in a thermostat at 20-25°C, and the culturing time is 16-36 hours.

6. The method of claim 5, wherein, Step (1) is as follows: obtaining the gene sequence of the alpha-amylase shown in SEQ ID NO. 2 by codon optimization of the amino acid sequence containing the alpha-amylase protein, performing gene synthesis, transfecting into a PET32a vector, and then transforming into BL21 bacteria, picking a single colony, inoculating into an LB culture medium containing Amp, and oscillating and culturing overnight; the next day, transferring the bacterial solution cultured overnight into a new LB culture medium containing Amp, culturing until the OD600 is 0.6-0.8, adding IPTG for oscillation induction, centrifuging the obtained mixed bacterial solution, ice-bath ultrasonic breaking, adding PBS buffer to resuspend the bacterial body, ultrasonic breaking, and centrifuging to obtain alpha-amylase protein precipitate.

7. The method of claim 5, wherein, Step (1) is: the picked single clone is inoculated in LB medium containing Amp, and is cultured in a shaking incubator overnight; the next day, the bacterial solution is transferred to LB medium containing Amp, and is cultured until the OD600 is 0.6-0.8, the number of colonies is not less than 10 6 CFU / mL to obtain the cultured bacterial solution; IPTG is added to the cultured bacterial solution to a final concentration of 0.5 mM, and after induction by oscillation, the precipitate is collected by centrifugation at 4000 rpm; then, ultrasonic crushing is performed under ice bath conditions, PBS buffer is added to resuspend the bacterial body, and after ultrasonic crushing at 4°C, centrifugation is performed at 15000 rpm to obtain one tube of α-amylase protein precipitate, which is stored at -20°C for later use.

8. The method according to any one of claims 5-7, characterized in that, Step (3) is as follows: filtering the kidney-shaped worm culture solution obtained in step (2) through a 0.45 μm sterile needle filter, retaining the supernatant filtrate, adding 2 mL of the filtrate into 1 tube of alpha-amylase protein precipitate to mix to obtain a 10× concentration alpha-amylase protein stimulating solution; different concentrations of alpha-amylase protein stimulating solutions can be prepared by dilution or concentration.

9. The method of claim 8, wherein, The concentration of the alpha-amylase protein stimulating solution is 5×-100×.

10. The method according to any of claims 5-7 or 9, characterized by, Step (4) is: adding 600 μL of the α-amylase protein stimulating solution obtained in step (3) into every 3 mL of the new nassula culture solution obtained in step (2) and mixing, and then placing in a constant temperature incubator at 25°C for culture, and counting the cell density every 3 hours with a plankton counting plate, drawing a growth curve and calculating the rate of reproductive cysts.

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