Genetically engineered bacterium for improving antiviral and immunocompetence of aquatic products as well as preparation method and application of genetically engineered bacterium

Abstract

The invention relates to a genetically engineered bacterium for improving the antiviral and immunocompetence of aquatic products and a preparation method and application thereof, the genetically engineered bacterium expresses novel C-type lectin PcLT of procambarus clarkia, and the amino acid sequence of the novel C-type lectin PcLT of procambarus clarkia comprises a sequence as shown in SEQ ID NO.1. The amino acid sequence of the novel C-type lectin PcLT of procambarus clarkia comprises a sequence as shown in SEQ ID NO.2. The starting strain of the genetically engineered bacterium is lactococcus lactis MG1363. According to the invention, a main antigen structure functional region of PcLT is subjected to recombinant expression in lactococcus lactis, the expression efficiency is high, and the lactococcus lactis is used as an expression vector, so that no toxic effect is generated, the balance of host intestinal flora can be promoted, the growth can be promoted, and the water quality can be improved.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a genetic engineering bacterium for improving the antiviral and immune capabilities of aquatic products, and a preparation method and application thereof. Background Art

[0002] Aquatic invertebrates lack a strong adaptive immune system like vertebrates, and they mainly rely on innate immunity to resist the invasion of pathogenic microorganisms. When invertebrates are infected by pathogenic microorganisms, their body fluids will secrete a variety of active substances, which on the one hand improve their own immunity, and on the other hand participate in a variety of activities to resist pathogenic microorganisms. Among the various body fluid active substances, enzyme activities such as superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP) are often used as reference indicators to measure the level of invertebrate immune activity.

[0003] The new C-type lectin PcLT of the shrimp is an important humoral immune factor that can improve the host's immunity and resist multiple viral infections. It has a broad-spectrum disease resistance. PcLT can not only help the body resist viral invasion, but also quickly eliminate bacteria in mixed infections. It can protect against attacks by common bacteria and viruses in aquaculture. Lactococcus lactis is a common probiotic with the advantages of high safety, easy cultivation, and regulation of intestinal flora balance. The competent state of Lactococcus lactis also has a good transformation and expression system, making it an ideal carrier for studying oral vaccines.

[0004] CN110041421A discloses a C-type lectin gLecB gene of Procambarus clarkii and the gLecB protein encoded by it, and further discloses the gLecB gene sequence and amino acid sequence. The expression vector is pGEX-5X-1-B-Lectin. A new C-type lectin gLecB in Procambarus clarkii is isolated and has antiviral effect.

[0005] So far, there is no genetically engineered Lactococcus lactis bacteria that highly expresses C-type lectin of Cambrian shrimp. How to significantly improve the protein expression efficiency and enhance the antiviral and antibacterial abilities of shrimp has become one of the urgent problems to be solved in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a genetically engineered bacterium for improving the antiviral and immune capabilities of aquatic products, and a preparation method and application thereof. The genetically engineered bacterium provided by the present invention can improve the antiviral and autoimmune capabilities of shrimp, making up for the defects of lack of specific immunity and insufficient nonspecific immunity.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a genetically engineered bacterium for improving antiviral and immune capabilities of aquatic products, wherein the genetically engineered bacterium expresses a novel C-type lectin PcLT of Procambarus clarkii, the amino acid sequence of the novel C-type lectin PcLT of Procambarus clarkii includes the sequence shown in SEQ ID NO.1, and the starting strain of the genetically engineered bacterium is Lactococcus lactis MG1363.

[0009] SEQ ID NO. 1: MNVEAFLLLLGSVLVYGDSPINKARYPAADITCPLPYKA VGGRCILFAVAEAGAWHDMGYFCETLGGKLVVIDDSTFMEALTDYILDGEFS TTEFWIGATDEVTEGVWKWVNGKTVRMRMPLWLTCSDDQEPNGGSAENCL AITSDHNYYFVDADCALAMNPICEYTVL.

[0010] The present invention recombinantly expresses PcLT in Lactococcus lactis with high expression efficiency. Lactococcus lactis is used as an expression vector, which has no toxic effect, can promote the balance of host intestinal flora, promote growth, and improve water quality.

[0011] Preferably, the nucleic acid sequence of the novel C-type lectin PcLT of Procambarus clarkii includes the sequence shown in SEQ ID NO.2.

[0012] SEQ ID NO.2: ATGAATGTCGAAGCTTTTCTTTTATTACTTGGCTCCGTT TTAGTATATGGAGATTCCCCTATCAATAAGGCAAGATATCCGGCAGCAGATATTACTTGCCCGCTCCCATACAAAGCTGTAGGAGGACGCTGTATTCTTTTTGCGGTCGCAGAAGCTGGTGCTTGGCATGATATGGGCTATTTTTGCGAAACCCTCGGCGGAAAATTAGTCGTCATTGATGACTCAACGTTTATGGAGGCATTGACCGATTACATCCTGGATGGTGAGTTTTCCAC CACGGAATTCTGGATTGGAGCTACAGACGAAGTTACTGAAGGGGTCTGGAAATGGGTTAACGGAAAAACGGTTCGTATGCGCATGCCTCTGTGGCTGACATGTTCTGATGATCAGGAACCGAACGGAGGGTCTGCGGAGAATTGCTTGGCAATAACCTCTGACCACAACTACTTTGTGGATGCAGATTGTGCCCTGGCTATGAACCCGATCTGTGAATATACCGTGCTCTAA.

[0013] Preferably, the novel C-type lectin PcLT of Procambarus clarkii is expressed in the genetically engineered bacteria via a recombinant plasmid.

[0014] Preferably, the starting plasmid of the recombinant plasmid is pLL32a plasmid, and the nucleic acid sequence of the pLL32a plasmid includes the sequence shown in SEQ ID NO.3.

[0015]

[0016] In a second aspect, the present invention provides a method for preparing a genetically engineered bacterium as described in the first aspect, the method for preparing the genetically engineered bacterium comprising: enzymatically ligating a gene expressing a novel C-type lectin PcLT of Procambarus clarkii with a pLL32a vector plasmid to obtain a recombinant plasmid, transforming the recombinant plasmid into competent Lactococcus lactis MG1363, inducing expression of PcLT, and obtaining the genetically engineered bacterium.

[0017] Preferably, the method of inducing expression comprises: anaerobic culture to OD 600 The pH value is 0.2 to 0.4 (for example, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38 or 0.4, etc.), and sake lactobacillus indica (SppIP) is added to induce the expression of the recombinant protein.

[0018] Preferably, the temperature of the anaerobic culture is 28-32°C (for example, 28°C, 29°C, 30°C, 31°C or 32°C, etc.).

[0019] Preferably, the final concentration of sake lactobacillus after addition is 40-60 ng / mL (for example, 40 ng / mL, 45 ng / mL, 48 ng / mL, 50 ng / mL, 52 ng / mL, 55 ng / mL or 60 ng / mL, etc.).

[0020] Preferably, after adding sake lactobacillus, the step of anaerobic culture at 28-32°C (for example, 28°C, 29°C, 30°C, 31°C or 32°C, etc.) for 7-9h (for example, 7h, 7.5h, 7.8h, 8h, 8.2h, 8.5h or 9h, etc.) is also included.

[0021] Preferably, the preparation method of the genetically engineered bacteria specifically comprises the following steps:

[0022] (1) The gene expressing PcLT and the pLL32a vector plasmid were double-digested, and the digestion products were separated by agarose gel electrophoresis. The PcLT target band and the pLL32a vector band were recovered by gel excision;

[0023] (2) PcLT was connected to pLL32a using ligase, and electroporated into competent Lactococcus lactis MG1363, and selected using ampicillin to obtain the recombinant plasmid pLL32a-PcLT;

[0024] (3) The recombinant Lactococcus lactis containing the recombinant plasmid was inoculated into MRS liquid medium containing ampicillin, and cultured at 28-32°C under anaerobic conditions for about 10-14 h. Then, the recombinant Lactococcus lactis containing the recombinant plasmid was inoculated into MRS liquid medium containing ampicillin at a volume ratio of 1:(80-120), and cultured at 28-32°C under anaerobic conditions until the OD 600 is 0.2-0.4, add sake lactobacillus to a final concentration of 40-60 ng / mL, and culture anaerobically at 28-32°C for 7-9 hours.

[0025] The specific point value of 28-32°C can be selected from 28°C, 29°C, 30°C, 31°C or 32°C, etc.

[0026] The specific point values ​​of the above 10 to 14 hours can be selected as 10h, 11h, 11.5h, 12h, 12.5h, 13h or 14h, etc.

[0027] The specific point values ​​of the above (80-120) can be selected as 80, 90, 95, 100, 105, 110, 120, etc.

[0028] The specific point values ​​of 0.2 to 0.4 mentioned above can be selected from 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38 or 0.4, etc.

[0029] The specific point values ​​of 40 to 60 ng / mL mentioned above can be selected from 40 ng / mL, 45 ng / mL, 48 ng / mL, 50 ng / mL, 52 ng / mL, 55 ng / mL or 60 ng / mL, etc.

[0030] The specific point values ​​of 7 to 9 hours mentioned above may be 7h, 7.5h, 8h, 8.5h or 9h etc.

[0031] In a third aspect, the present invention provides a recombinant protein, wherein the preparation method of the recombinant protein comprises:

[0032] The genetically engineered bacteria described in the first aspect are cultured, the cells are disrupted and the PcLT protein is purified to obtain the recombinant protein.

[0033] In a fourth aspect, the present invention provides a composite probiotic for improving antiviral and immune capabilities of aquatic products, wherein the composite probiotic comprises the genetically engineered bacteria described in the first aspect and Bacillus subtilis subsp. subtilis CICC 6060.

[0034] The present invention prepares a composite probiotic by compounding a genetically engineered bacterium that induces expression of PcLT protein with Bacillus subtilis subsp. subtilis CICC 6060. The two bacteria synergistically prevent and treat shrimp white spot virus and enhance shrimp immunity, and the composite probiotic has broad application prospects.

[0035] Preferably, the bacterial count ratio of the genetically engineered bacteria to Bacillus subtilis subsp. subtilis is 1:(0.5-2).

[0036] The specific point values ​​of the above (0.5-2) can be selected from 0.5, 0.7, 0.9, 1, 1.1, 1.3, 1.5, 1.7 or 2, etc.

[0037] In a fifth aspect, the present invention provides the use of any one of the genetically engineered bacteria described in the first aspect, the preparation method of the genetically engineered bacteria described in the second aspect, the recombinant protein described in the third aspect, or the composite probiotics described in the fourth aspect in the preparation of a drug for preventing and treating white spot virus in shrimp and improving the immune activity of shrimp.

[0038] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) The present invention optimizes the main antigenic structural functional region of the novel C-type lectin PcLT of Procambarus clarkii, and then recombinantly expresses it in Lactococcus lactis with high expression efficiency. Using Lactococcus lactis as an expression vector has no toxic effects, can promote the balance of the host intestinal flora, promote growth, and improve water quality.

[0041] (2) The genetically engineered bacteria prepared by the present invention and Bacillus subtilis subsp. subtilis can synergistically prevent and treat white spot virus in shrimp, so that the diseased shrimp can quickly recover, eat normally, and the number of virus copies can be significantly reduced. The immune ability of shrimp can be quickly improved, and the activities of SOD, ACP, and AKP in shrimp can be increased, which makes up for the defects of lack of specific immunity and insufficient nonspecific immunity of aquatic invertebrates and enhances the humoral effect in the nonspecific immunity of shrimp. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a diagram showing the results of double enzyme digestion electrophoresis identification of the PcLT gene and pLL32a plasmid in Example 1.

[0043] Figure 2 The electrophoresis results of the recombinant PcLT proteins of Example 2 and Comparative Example 1 are shown. DETAILED DESCRIPTION

[0044] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0045] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0046] The Lactococcus lactis used in the following examples and test examples is Lactococcus lactis MG1363, and the Bacillus subtilis subsp. subtilis is Bacillus subtilis subsp. subtilis CICC 6060.

[0047] Example 1

[0048] This example constructs a recombinant plasmid expressing a novel C-type lectin PcLT of Procambarus clarkii and prepares a genetically engineered bacterium for improving the antiviral and immune capabilities of aquatic products, comprising the following steps:

[0049] (1) The nucleotide sequence of the novel C-type lectin PcLT of Procambarus clarkii was found in NCBI, and its main antigenic structural functional region was optimized, as shown in SEQ ID NO. 2. The sequence was sent to Shanghai Biotechnology Co., Ltd. for synthesis.

[0050] (2) The PcLT gene and the pLL32a vector plasmid were double-digested. The nucleic acid sequence of the pLL32a vector plasmid is shown in SEQ ID NO.3. The digestion products were separated by agarose gel electrophoresis. The electrophoresis results were as follows: Figure 1 As shown, the PcLT target band and the pLL32a vector band were recovered by gel excision, and PcLT and pLL32a were connected with ligase.

[0051] (3) Use a pre-cooled pipette tip to draw up 10 μL of the ligation product, gently stir it, and add it to 100 μL of pre-cooled Lactococcus lactis competent cells MG1363. Then, transfer the mixed bacterial solution into a pre-cooled 0.1 cm sterile electroporation cup, place it on ice for 5 minutes, and then perform electroporation.

[0052] (4) Set the electroporation conditions of the electroporator: 2000 V, 400 Ω, 25 μF, and perform one pulse.

[0053] (5) After the reaction is complete, the liquid in the electroporation cup is aspirated into a centrifuge tube and 600 μL of pre-cooled MRS recovery liquid medium containing sucrose is immediately added.

[0054] (6) Place on ice for 10 min and then incubate anaerobically at 30°C for 3 h.

[0055] (5) Take 200 μL of the bacterial solution and spread it on an MRS agar plate containing ampicillin. Culture it anaerobically at 30°C. The single colony obtained is the colony containing the recombinant plasmid pLL32a-PcLT.

[0056] Example 2

[0057] The present embodiment provides a method for inducing genetically engineered bacteria to express recombinant proteins, comprising the following steps: inoculating the genetically engineered bacteria prepared in Example 1 into an MRS liquid culture medium containing ampicillin, culturing at a constant temperature of about 12 h under anaerobic conditions at 30°C, inoculating into a fresh MRS liquid culture medium containing ampicillin at a volume ratio of 1:100, and culturing at a constant temperature of 30°C under anaerobic conditions until the OD 600 When the pH value was 0.3, SppIP (final concentration was 50 ng / mL) was added to induce the culture. The culture was stopped at 30°C under anaerobic conditions for 8 h, and the bacteria were collected by centrifugation at 2000 rpm for 20 min.

[0058] Example 3

[0059] This embodiment provides a method for inducing genetically engineered bacteria to express recombinant proteins, which is different from Embodiment 2 only in that the genetically engineered bacteria are cultured under aerobic conditions.

[0060] Example 4

[0061] This example provides a method for inducing genetically engineered bacteria to express recombinant proteins, which is different from Example 2 only in that the final concentration of sake lactobacillus is 40 ng / mL.

[0062] Example 5

[0063] This example provides a method for inducing genetically engineered bacteria to express recombinant proteins, which is different from Example 2 only in that the final concentration of sake lactobacillus is 60 ng / mL.

[0064] Example 6

[0065] In this example, a composite probiotic was prepared. The expression of recombinant protein was induced using the method provided in Example 2 to obtain genetically engineered bacteria expressing PcLT, which were mixed with Bacillus subtilis subsp. subtilis. The bacterial count ratio of the genetically engineered bacteria to Bacillus subtilis subsp. subtilis was 1:1.

[0066] Example 7

[0067] This example prepares a composite probiotic, which differs from Example 6 only in that it does not contain the genetically engineered bacteria expressing PcLT, and is replaced by Bacillus subtilis subsp. subtilis with an equal bacterial count.

[0068] Example 8

[0069] This example prepares a composite probiotic, which differs from Example 6 only in that it does not contain Bacillus subtilis subsp. subtilis and is replaced by an equal number of genetically engineered bacteria expressing PcLT.

[0070] Example 9

[0071] This example prepares a composite probiotic, which differs from Example 6 only in that the genetically engineered bacteria expressing PcLT are replaced with empty Lactococcus lactis MG1363 bacteria with an equal number of bacteria.

[0072] Example 10

[0073] This example prepares a composite probiotic, which is different from Example 6 only in that Bacillus subtilis subsp. subtilis is replaced by Bacillus coagulans with an equal bacterial count.

[0074] Embodiment 11

[0075] This example prepares a composite probiotic, which is different from Example 6 only in that the bacterial count ratio of the genetically engineered bacteria to Bacillus subtilis subsp. subtilis is 1:2.

[0076] Example 12

[0077] This example prepares a composite probiotic, which is different from Example 6 only in that the bacterial count ratio of the genetically engineered bacteria to Bacillus subtilis subsp. subtilis is 1:0.5.

[0078] Comparative Example 1

[0079] This comparative example constructs a recombinant plasmid expressing a novel C-type lectin PcLT of Procambarus clarkii and prepares a genetically engineered bacterium for improving the antiviral and immune capabilities of aquatic products, and induces the genetically engineered bacterium to express the recombinant protein, comprising the following steps:

[0080] (1) pLL32a-PcLT and pET32a plasmids were double-digested, and the digestion products were separated by agarose gel electrophoresis. The PcLT band and the pET32a vector band were recovered by gel excision, ligated with T4 ligase, transformed into BL21 (DE3) competent cells, and screened with 0.5 μg / mL ampicillin to obtain recombinant plasmid pET32a-PcLT and recombinant Escherichia coli.

[0081] (2) The recombinant bacteria were cultured in LB liquid medium containing 0.5 μg / mL ampicillin with shaking at 37°C overnight.

[0082] (3) Take the overnight culture solution and inoculate it into LB medium to make the OD 600The mixture was cultured at 37℃ with shaking for about 2 hours until the OD 600 When the concentration of IPTG reaches about 0.6, add IPTG inducer, and the final concentration of IPTG is 1mM.

[0083] (4) After 8 h of induction, the fermentation was terminated and the cells were collected by centrifugation at 2000 rpm for 20 min.

[0084] Test Example 1

[0085] In this test example, the genetically engineered bacteria expressing PcLT prepared in Examples 2-5 and Comparative Example 1 were broken and purified to obtain recombinant PcLT protein. The recombinant PcLT protein was purified by ion exchange and hydrophobic chromatography. The protein concentration was determined by the BCA method. The operation steps were performed according to the Bio-Tech BCA protein concentration determination kit (P0012). The expression of the PcLT protein was measured as shown in Table 1. The electrophoresis results of the recombinant PcLT protein obtained in Example 2 and Comparative Example 1 are shown in Table 1. Figure 2 shown.

[0086] Table 1

[0087]

[0088]

[0089] It can be seen from Table 1 that:

[0090] (1) Comparing Example 2 with Comparative Example 1, it can be seen that using Lactococcus lactis as a strain for expressing PcLT protein has a higher expression efficiency than Escherichia coli.

[0091] (2) Comparing Example 2 with Example 3, it can be seen that Example 2 is an anaerobic condition, while Example 3 is an aerobic condition. The anaerobic condition is more suitable for the expression of PcLT protein.

[0092] (3) Comparing Example 2 with Example 4 and Example 5, it can be seen that the difference lies in the different concentrations of the inducer SppIP. Too high or too low a concentration will affect the expression of PcLT protein.

[0093] Test Example 2

[0094] In this test example, 10 mL of the composite probiotics prepared in Examples 6-12 were taken, mixed evenly with 8 catties of shrimp feed, and fed to 100 sick shrimps infected with white spot virus. Ten shrimps were randomly selected before feeding, 24h, 48h and 72h after feeding, and the average values ​​of the virus copy number, SOD activity, ACP activity and AKP activity in the hepatopancreas were measured. The shrimp feed without adding composite probiotics was used to feed the sick shrimp infected with white spot virus as a blank control. The determination method is as follows: the virus copy number is measured using the fluorescent quantitative PCR method, and the SOD, ACP, and AKP activities are all measured using the Nanjing Jiancheng kit. The specific determination is carried out with reference to the kit instructions, and the test results are shown in Table 2.

[0095] Table 2

[0096]

[0097]

[0098]

[0099] It can be seen from Table 2 that:

[0100] (1) Comparing Example 6 with the blank control group, it can be seen that after feeding with the composite probiotics, the diseased shrimps basically improved, with only a few shrimps swimming in the pond, but eating normally, and the number of viral copies showed a downward trend. The 72h test result was negative, indicating that the composite bacteria has the effect of preventing and treating white spot virus in shrimp; SOD activity, ACP activity and AKP activity are at their maximum at 24h, and then decrease due to body consumption, indicating that the composite bacteria has the ability to improve shrimp immunity.

[0101] (2) Comparing Example 6 with Examples 7-10, it can be seen that the genetically engineered bacteria and Bacillus subtilis subsp. subtilis have a synergistic effect, and the lack or replacement of any one of the bacteria will reduce the antiviral ability of the composite bacteria.

[0102] (3) Comparing Example 6 with Examples 11-12, it can be seen that the ratio of genetically engineered bacteria to Bacillus subtilis subsp. subtilis also has a certain influence on the effect of the composite bacteria in preventing and controlling shrimp white spot virus and improving the immune activity of shrimp.

[0103] In summary, the present invention recombinantly expresses PcLT in Lactococcus lactis, with high expression efficiency. Lactococcus lactis is used as an expression vector, which has no toxic effects and can promote the balance of the host intestinal flora and promote growth. The present invention also prepares a composite probiotic by compounding the prepared genetically engineered bacteria with Bacillus subtilis subtilis, which has the effect of synergistically preventing and treating shrimp white spot virus and improving shrimp immune activity.

[0104] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A genetically engineered bacterium for improving the antiviral and immune capabilities of aquatic products, characterized in that: The genetically engineered bacteria expresses a novel C-type lectin PcLT of Procambarus clarkii, the amino acid sequence of the novel C-type lectin PcLT of Procambarus clarkii includes the sequence shown in SEQ ID NO.1, and the starting strain of the genetically engineered bacteria is Lactococcus lactis MG1363.

2. The genetically engineered bacterium according to claim 1, characterized in that: The nucleic acid sequence of the novel C-type lectin PcLT of Procambarus clarkii includes the sequence shown in SEQ ID NO.2; Preferably, the novel C-type lectin PcLT of Procambarus clarkii is expressed in the genetically engineered bacteria via a recombinant plasmid.

3. The genetically engineered bacterium according to claim 2, characterized in that: The starting plasmid of the recombinant plasmid is pLL32a plasmid, and the nucleic acid sequence of the pLL32a plasmid includes the sequence shown in SEQ ID NO.

3.

4. A method for preparing a genetically engineered bacterium according to any one of claims 1 to 3, characterized in that: The preparation method of the genetically engineered bacteria comprises: enzymatically connecting the gene expressing the novel C-type lectin PcLT of Procambarus clarkii with the pLL32a vector plasmid to obtain a recombinant plasmid, transforming the recombinant plasmid into competent Lactococcus lactis MG1363, inducing the expression of PcLT, and obtaining the genetically engineered bacteria.

5. The method for preparing the genetically engineered bacteria according to claim 4, characterized in that: The method of inducing expression comprises: anaerobic culture to OD 600 is 0.2-0.4, and sake lactobacillus is added to induce the expression of recombinant protein; Preferably, the temperature of the anaerobic culture is 28-32°C; Preferably, the final concentration of the sake lactobacillus after addition is 40-60 ng / mL; Preferably, after adding sake lactobacillus, the method further comprises the step of anaerobic culture at 28-32° C. for 7-9 hours.

6. The method for preparing the genetically engineered bacteria according to claim 4 or 5, characterized in that: The preparation method of the genetically engineered bacteria specifically comprises the following steps: (1) The gene expressing PcLT and the pLL32a vector plasmid were double-digested, and the digestion products were separated by agarose gel electrophoresis. The PcLT target band and the pLL32a vector band were recovered by gel excision; (2) PcLT was connected to pLL32a using ligase, and electrotransformed into competent Lactococcus lactis MG1363, and selected using ampicillin to obtain the recombinant plasmid pLL32a-PcLT; (3) The recombinant Lactococcus lactis containing the recombinant plasmid was inoculated into MRS liquid medium containing ampicillin, and cultured at 28-32°C under anaerobic conditions for about 10-14 h. Then, the recombinant Lactococcus lactis containing the recombinant plasmid was inoculated into MRS liquid medium containing ampicillin at a volume ratio of 1:(80-120), and cultured at 28-32°C under anaerobic conditions until the OD 600 is 0.2-0.4, add sake lactobacillus to a final concentration of 40-60 ng / mL, and culture anaerobically at 28-32°C for 7-9 hours.

7. A recombinant protein, characterized in that The method for preparing the recombinant protein comprises: The genetically engineered bacteria according to any one of claims 1 to 3 are cultured, the cells are disrupted and the PcLT protein is purified to obtain the recombinant protein.

8. A composite probiotic for improving the antiviral and immune capabilities of aquatic products, characterized in that: The composite probiotics comprises the genetically engineered bacteria according to any one of claims 1 to 3 and Bacillus subtilis subsp. subtilis CICC 6060.

9. The composite probiotic according to claim 8, characterized in that: The bacterial count ratio of the genetically engineered bacteria to Bacillus subtilis subsp. subtilis is 1:(0.5-2).

10. Use of any one of the genetically engineered bacteria according to any one of claims 1 to 3, the preparation method of the genetically engineered bacteria according to any one of claims 4 to 6, the recombinant protein according to claim 7 or the composite probiotics according to claim 8 or 9 in the preparation of drugs for preventing and treating white spot virus in shrimp and improving shrimp immune activity.

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