Firefly luciferase-labeled Talaromyces marneffei strain and construction method

Abstract

The present invention discloses a firefly luciferase-labeled Talaromyces marneffei strain and a construction method, the strain is named TM-LUC strain, and its microbial preservation number is: CGMCC No.40152; the firefly luciferase-labeled Talaromyces marneffei strain and the construction method of the present invention include: S1, constructing a firefly luciferase recombinant plasmid; S2, transferring into the genome of TM. The present invention transfers firefly luciferase into the TM genome, so that TM can encode luciferase, and bioluminescence is performed under the action of substrate luciferin, replacing the traditional ATCC18224 strain. After TM-LUC infects animals, intraperitoneal injection of luciferin substrate can theoretically make the strain glow, thereby realizing the monitoring of strain distribution in the animal body; the treatment process after administration can also be monitored; and a new direction is provided for the exploration of latent infection mechanisms.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing a firefly luciferase-labeled Talaromyces marneffei strain. Background Art

[0002] Talaromyces marneffei (TM) is an opportunistic pathogen and the only known pathogenic fungus in the genus Talaromyces. It is highly prevalent in immunodeficiency patients. TM infection is a regional infectious disease, mainly prevalent in AIDS populations in southern China and ASEAN countries. In Guangxi, the mortality risk of AIDS combined with TM infection is higher than that of other opportunistic infections (AHR = 4.51). Due to its high morbidity and mortality, TM infection has become a serious public health problem in Southeast Asia and southern China.

[0003] In clinical practice, patients with TM infection are often treated with amphotericin B, which is more effective than traditional itraconazole. However, the efficacy has not been very ideal. Many clinical case reports show that after the initial cure of TM, if the medication is not taken within a period of time, there is a risk of recurrence. This suggests that TM may have a mature immune escape strategy in the body, and it can remain dormant in the body and relapse under certain conditions. However, little is known about the mechanism of TM latent reinfection, including the location of TM latent infection and the mechanism of relapse. The main reason is the lack of a suitable TM infection visualization model. The traditional TM-mouse infection model has the following disadvantages: (1) Mice need to be slaughtered at different time points and different organs need to be cultured for fungal culture to obtain bacterial load data, so a large number of mice are required; (2) Since the TM culture positive rate is only 70-80%, most of the latent TM cannot be cultured due to the low bacterial load, so it is impossible to determine its latent location; (3) Due to individual differences, the infection process of different batches or different mice in the same batch is different, which will cause experimental errors. The main reason for the above shortcomings is the lack of firefly luciferase-labeled T. marneffei. There is also a lack of relevant research at home and abroad.

[0004] In addition, bioluminescence imaging (BLI) is widely used in the biomedical field due to its non-invasive, quantitative analysis, and high sensitivity. Among them, the most in-depth research is firefly luciferase. The firefly luciferase reporter system is often a firefly luciferase / D-luciferin substrate. The D-luciferin substrate is converted into an activated state of oxidized luciferin under the catalysis of firefly luciferase, and it emits bioluminescence in the process. The luciferin imaging visualization model can track the diffusion process of the same observation target by recording the same group of experimental subjects at different points, so the data obtained is more authentic and reliable. In addition, the luminescence system has high detection sensitivity and its penetration can reach 3-4cm. It can detect organs deeper in the mouse body (the mouse body thickness is about 7-8cm), and as long as the total number of cells reaches more than 100, the signal can be detected. Therefore, this technology has been widely used in monitoring tumor burden or treatment effects in animal models. In fungal research, imaging of Candida albicans encoding firefly luciferase in mice revealed its invisible infection site, which brought new directions for the treatment of Candida albicans and explained to some extent the reason for the recurrence of Candida albicans. This shows the wide applicability of firefly luciferase.

[0005] TM is a common fungus of HIV opportunistic infection, and its high recurrence rate and mortality rate seriously affect the prognosis of AIDS population. Therefore, the development of therapeutic drugs for TM should require more and more in-depth research. This application proposes to construct Talaromyces marneffei labeled with firefly luciferase, laying a solid foundation for the construction of subsequent TM infection visualization model. Firefly luciferase-labeled TM will add bioluminescence technology to traditional basic research, which will bring new directions for TM animal model research and make it possible to monitor the infection site and treatment effect of TM in animals. Summary of the invention

[0006] To this end, this application provides the following technical solutions:

[0007] A firefly luciferase-labeled Talaromyces marneeffei strain, named TM-LUC strain, was deposited in the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its microbial accession number is: CGMCC No.40152, and the deposit date is March 25, 2022.

[0008] A method for constructing a firefly luciferase-labeled Talaromyces marneffei strain, comprising:

[0009] S1, construction of firefly luciferase recombinant plasmid;

[0010] S2, genome transferred into TM.

[0011] The step S1 includes plasmid construction, specifically:

[0012] S11, luciferase fragment amplification, using pGL3-basic plasmid as template, design primers and amplify luciferase fragment;

[0013] pCAMBIA1303-TrpC-Hygro-gpdA-GFP vector backbone was digested with S12, NcoI, and BstEII;

[0014] S13, recombination and ligation, recombining and ligating the recovered PCR product (luciferase fragment) with the pCAMBIA1303-TrpC-Hygro-gpdA-GFP vector backbone recovered after enzyme digestion;

[0015] S14, identification of recombination products: select several clones on the recombination reaction transformation plate for colony PCR identification.

[0016] The step S2 comprises:

[0017] S21. Agrobacterium transformation culture, including:

[0018] 210. Take EHA105 competent cells, add plasmid DNA (pCAMBIA1303-TrpC-Hygro-gpdA-Luc), and mix well;

[0019] 211. Add LB liquid medium without antibiotics and culture at 28°C with shaking for 2 to 3 hours;

[0020] 212. Collect the bacteria by centrifugation, keep the supernatant, gently blow and resuspend the bacteria, and spread it on the LB plate containing the corresponding antibiotics. Invert and place it in a 28℃ incubator for 2-3 days. Pick the positive clones and transfer them to LB medium and shake at 28℃.

[0021] S22, Agrobacterium infection with Talaromyces marnii, including:

[0022] 221. Add 1% of EHA105 bacterial solution containing plasmid (pCAMBIA1303-TrpC-Hygro-gpdA-Luc) into TM and culture with shaking at 28°C for 24 h.

[0023] 222. Add glass beads to the bacterial solution after co-culture, shake to break up the clumped bacteria, then spread on a solid Sabouraud glucose medium plate containing hygromycin, and culture in a 37°C incubator until a single clone grows out;

[0024] 223. The monoclonal transformant was transferred to a new liquid Sabouraud medium containing hygromycin, cultured in a 37°C incubator with shaking until the logarithmic growth phase, and the genome was extracted for identification.

[0025] The firefly luciferase-labeled Talaromyces marneffei strain was constructed using the above method.

[0026] The use of the firefly luciferase-labeled Talaromyces marneffei strain in the preparation of a drug for preventing or treating TM infection.

[0027] This application has the following advantages:

[0028] 1) Construction of a firefly luciferase-labeled T. marneffei strain, transferring firefly luciferase into the TM genome so that TM can encode luciferase and emit bioluminescence under the action of the substrate luciferin, which will make real-time fluorescence imaging of animal models and monitoring of the treatment process feasible.

[0029] 2) The use of firefly luciferase-labeled T. marneffei strains replaces the traditional ATCC18224 strain. After TM-LUC infects animals, intraperitoneal injection of luciferin substrate can theoretically make the strain glow, thereby monitoring the strain distribution in the animal body; it can also monitor the treatment process after drug administration, providing a new direction for the exploration of latent infection mechanisms.

[0030] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application so that it can be implemented in accordance with the contents of the specification, and to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the following is a detailed description of the preferred embodiments of the present application in conjunction with the accompanying drawings as follows.

[0031] Based on the detailed description of the specific embodiments of the present application in combination with the accompanying drawings below, those skilled in the art will become more aware of the above and other objects, advantages and features of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without creative work. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale.

[0033] Figure 1Construction and verification of firefly luciferase plasmid and transformation products, A. pCAMBIA1303-TrpC-Hygro-gpdA-GFP plasmid map; B. pCAMBIA1303-TrpC-Hygro-gpdA-Luc plasmid map; C. Verification of TM-LUC, lane 1: 18224 wild control amplification product; lane 2: trans2kplusII DNA marker; lanes 3-5: amplification products of transformants 1-3; D. pCAMBIA1303-TrpC-Hygro-gpdA-Luc plasmid electrophoresis results (lane 7);

[0034] Figure 2 is a screening line graph of the sensitivity of Talaromycin to marneffei;

[0035] Figure 3 is a schematic diagram of PCR identification of Agrobacterium transformed TM;

[0036] Figure 4 Figure 1 is the growth of TM-WT and TM-LUC, A. Average growth curve (p=0.647) and average growth rate curve (p=0.912) in PDA medium at 27°C; B. Average growth curve (p=0.619) and average growth rate curve (p=0.08) in BHI agar medium at 37°C; C. Cotton blue staining morphology of TM-WT and TM-LUC on the 16th day, top: TM-WT, bottom: TM-LUC; D. Growth of TM-WT and TM-LUC on PDA and BHI medium on the 14th day, from left to right, respectively, WT front, WT back, LUC front, LUC back, top: 27°C, PDA medium, bottom: 37°C, BHI medium;

[0037] Figure 5 It is the detection of verification factors and cell death of THP-1 macrophages infected with TM-WT or TM-LUC; A. Detection of inflammatory factors of THP-1 macrophages infected with TM-WT or TM-LUC for 24h and 48h; B. Detection of cell death of THP-1 macrophages infected with TM-WT or TM-LUC for 48h and 72h;

[0038] Figure 6 TM-LUC luminescence detection and real-time fluorescence imaging of animals; A. Luminescence detection of luciferin substrate at different time points; B. Detection of TM-LUC fluorescence intensity by imaging instrument. DETAILED DESCRIPTION

[0039] To make the purpose, technical scheme and advantages of the embodiment of the present application clearer, the technical scheme in the embodiment of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present application. Obviously, the described embodiment is a part of the embodiment of the present application, rather than all of the embodiments. In the following description, specific details such as specific configuration and components are provided only to help fully understand the embodiments of the present application. Therefore, it should be clear to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, for clarity and brevity, the description of known functions and structures is omitted in the embodiment.

[0040] The term "and / or" in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist at the same time. The term " / and" in this article describes another type of association object relationship, indicating that there can be two relationships. For example, A / and B can mean: A exists alone, and A and B exist alone. In addition, the character " / " in this article generally indicates that the previous and next associated objects are in an "or" relationship.

[0041] It should also be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusions.

[0042] Example 1

[0043] A method for constructing a firefly luciferase-labeled Talaromyces marneffei strain comprises: S1, constructing a firefly luciferase recombinant plasmid; S2, transferring into a TM genome.

[0044] The strain is deposited in the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing. Its microbial preservation number is: CGMCC No.40152, and the preservation date is March 25, 2022.

[0045] Example 2

[0046] This example constructs a firefly luciferase recombinant plasmid.

[0047] (1) Plasmid construction

[0048] ① Luciferase fragment amplification. Using pGL3-basic plasmid as template, design primers and amplify the luciferase fragment (amplification primers see Table 1, amplification system see Table 2, reaction conditions see Table 3)

[0049] Table 1 Firefly luciferase primer sequences

[0050]

[0051] Table 2 Luciferase fragment amplification system

[0052] name Volume(ul) Q5 High Fidelity Enzyme 0.5 Q5 Buffer (5x) 10 dNTP(10mM) 2 Primer F 2 Primer R 2 Template (pGL3-basic plasmid diluted 100 times) 0.5 ddH2O 33

[0053] Table 3 Luciferase fragment amplification reaction conditions

[0054]

[0055]

[0056] ② The pCAMBIA1303-TrpC-Hygro-gpdA-GFP vector backbone was digested with NcoI and BstEII, reacted at 37°C for 1 hour, inactivated at 80°C for 10 minutes, and the vector backbone was recovered by 1.5% agarose gel electrophoresis (the digestion system is shown in Table 4, and the plasmid backbone was quantitatively loaded according to the concentration of different batches).

[0057] Table 4 Enzyme digestion of pCAMBIA1303-TrpC-Hygro-gpdA-GFP vector backbone system

[0058] name Volume(ul) Endonuclease NcoI 1 Endonuclease BstEII 1 Buffer(10x) 2 Plasmid backbone (1ug) Calculated based on plasmid concentration ddH2O To20

[0059] ③ Recombination ligation. The recovered PCR product (luciferase fragment) was recombined with the pCAMBIA1303-TrpC-Hygro-gpdA-GFP vector backbone recovered after enzyme digestion, reacted at 37°C for 30 minutes, and then quickly cooled on ice (see Table 5 for the recombination ligation system).

[0060] Table 5 Recombination system

[0061] name Volume(ul) Insert 1 Linearized vector 1 5×CE II buffer 2 Exnase II 1 ddH2O To 10

[0062] Then, transform the recombinant product. Thaw TranT1 competent cells on ice, take 10ul of the recombinant product and add it to 100ul of competent cells, gently tap the tube wall to mix (do not oscillate to mix), and let it stand on ice for 30min; after heat shock in a 42℃ water bath for 45s, immediately place it on ice to cool for 2-3min, add LB medium (without adding antibiotics), and shake the bacteria at 37℃ for 1h (speed 200-250rpm); preheat the LB (kanamycin 50ug / ml) solid culture medium plate with corresponding resistance in a 37℃ incubator, centrifuge at 5,000rpm for 5min, and discard 900ul of supernatant; resuspend the bacteria with the remaining culture medium, gently spread it on the plate containing kanamycin 50ug / ml with a sterile spreading stick, and invert and culture it in a 37℃ incubator for 12-16h.

[0063] ④ Identification of recombination products. Select several clones on the recombination reaction transformation plate for colony PCR identification. The primers are the same as those in Table 1, the amplification system is shown in Table 6, and the reaction conditions are shown in Table 7.

[0064] Table 6 Recombinant product identification system

[0065] name Volume(ul) 2×Taq Plus Master Mix Ⅱ(Dye Plus) 10 Primer F 1 Primer R 1 Bacterial liquid 1 ddH2O 7

[0066] Table 7 Recombinant product amplification reaction conditions

[0067]

[0068] like Figure 1 As shown, Figure 1 Construction and verification of firefly luciferase plasmid and transformation product, A. pCAMBIA1303-TrpC-Hygro-gpdA-GFP plasmid map; B. pCAMBIA1303-TrpC-Hygro-gpdA-Luc plasmid map; C. pCAMBIA1303-TrpC-Hygro-gpdA-Luc plasmid electrophoresis results (lane 7).

[0069] The pCAMBIA1303-TrpC-Hygro-gpdA-GFP vector backbone was digested with NcoI and BstEII, and the sequence between the two was replaced with the luciferase sequence (full length 1689 bp) to obtain the pCAMBIA1303-TrpC-Hygro-gpdA-Luc plasmid. Several clone colonies on the recombination reaction plate were selected for PCR identification. The electrophoresis results showed that the band appeared between 1000-2000 bp. The colony in the 7th lane was then expanded and cultured, and the plasmid was extracted for sequencing and comparison. The sequencing results were correct, proving that the pCAMBIA1303-TrpC-Hygro-gpdA-Luc plasmid was successfully constructed.

[0070] (2) Hygromycin concentration screening

[0071] like Figure 2 As shown, Figure 2 This is a screening of T. marneffei's sensitivity to hygromycin. T. marneffei was spread on Sabouraud glucose medium plates containing different concentrations of hygromycin (0μg / ml, 20μg / ml, 40μg / ml, 60μg / ml, 80μg / ml, 100μg / ml), cultured at 37°C for 3 days, and the lowest hygromycin concentration with no colony growth was screened out as the concentration for subsequent screening of T. marneffei transformants.

[0072] TM-WT did not grow at a hygromycin concentration of 80 μg / ml.

[0073] (3) Agrobacterium transformation culture

[0074] Take 100μl EHA105 competent cells and add 0.1μg plasmid DNA (pCAMBIA1303-TrpC-Hygro-gpdA-Luc), stir the bottom of the tube by hand to mix, and then stand on ice for 5 minutes, liquid nitrogen for 5 minutes, 37℃ water bath for 5 minutes, and ice bath for 5 minutes. Add 700μl LB liquid medium without antibiotics and shake at 28℃ for 2-3 hours. Centrifuge at 6000rpm for 1min to collect bacteria, keep about 100μl supernatant, gently blow and resuspend the bacteria, and apply it on the LB plate containing the corresponding antibiotics (50μg / ml kanamycin, 20μg / ml rifampicin), invert and place in a 28℃ incubator for 2-3 days, pick the positive clones and put them into 10ml LB (As20mg / L) medium, and shake at 28℃ until OD600 is about 0.8.

[0075] Example 3

[0076] In this example, Agrobacterium infection TM was performed.

[0077] Add EHA105 bacterial solution containing plasmid (pCAMBIA1303-TrpC-Hygro-gpdA-Luc) into TM at a ratio of 1%, and culture at 28°C with shaking for 24 h.

[0078] After adding glass beads to the co-cultured bacterial solution, the clumped bacteria were broken up by shaking, and then spread on a solid Sabouraud glucose medium plate containing 150μg / ml hygromycin, and cultured in a 37°C incubator until a single clone grew out (about 3-7 days). The single clone transformant was transferred to a new liquid Sabouraud medium containing 150μg / ml hygromycin, and cultured in a 37°C incubator until the logarithmic growth phase, and the genome was extracted for relevant identification.

[0079] RT-PCR was used to identify the transformed strains of T. marneffei. The mycelial blocks of T. marneffei were taken with a 1 ml pipette tip, ground with liquid nitrogen, and 1 ml of Trizol reagent was added. The mixture was blown and beaten with a pipette and transferred to a 1.5 ml EP tube without RNase. The tube was lysed for 10 min, 200 μl of chloroform was added, and the mixture was mixed by violent inversion several times. The tube was placed at room temperature for 5 min, and centrifuged at 4°C, 12,000 g for 15 min. The tube was visibly separated into three phases: upper (RNA), middle (protein), and lower (DNA). Transfer the upper aqueous phase (about 400μl) to another new 1.5ml EP tube, add 400μl isopropanol, mix well and let stand at room temperature for 10min, centrifuge at 4℃, 12000rpm for 10min, white RNA precipitate can be seen at the bottom of the tube, discard the supernatant, add 1ml of RNase-free 75% ethanol, vortex mix well, centrifuge at 4℃, 10000rpm for 5min, repeat the ethanol washing step once, discard the supernatant, dry the RNA precipitate in air for 5-10min, and dissolve the precipitate in 20μl DEPC water.

[0080] Take 2 μl of the dissolved RNA and measure OD260, OD280 and OD260 / OD280 values ​​with a micro-spectrophotometer to calculate the purity and concentration of the RNA. Reverse transcription of RNA into cDNA, the reaction conditions are shown in Tables 8 and 9 below.

[0081] ① Genomic DNA removal:

[0082] Table 8 Genomic DNA removal system

[0083]

[0084] ②Reverse transcription reaction:

[0085] Table 9 Reverse transcription reaction system

[0086]

[0087] Reaction conditions: 37℃15min, 85℃5s

[0088] Luciferase was amplified by PCR and then verified by electrophoresis. The primer sequences (Table 10), amplification system (Table 11) and reaction conditions (Table 12) used were as follows.

[0089] Table 10 Primer sequences

[0090]

[0091] Table 11 Amplification system

[0092] name Volume(ul) 2×Taq Plus Master Mix Ⅱ(Dye Plus) 10 Primer F 1 Primer R 1 cDNA 1 ddH2O 7

[0093] Table 12 Reaction conditions

[0094]

[0095]

[0096] like Figure 3 As shown, Figure 3 Schematic diagram of PCR identification of Agrobacterium transformed TM.

[0097] The full length of the luciferase sequence used in PCR was 636 bp, and the electrophoresis results showed that the TM-LUC band appeared between 500-750 bp, proving that the TM-LUC transformant was successfully constructed.

[0098] Example 4

[0099] TM-LUC and TM-WT were expanded and cultured in PDA medium and BHI medium.

[0100] Specifically,

[0101] (1) The expansion culture of TM-WT and TM-LUC includes:

[0102] TM strain: ATCC18224 strain, ATCC18224-LUC strain

[0103] TM-WT and TM-LUC expansion culture: Take 8.02g potato dextrose agar powder, add it to 200ml ultrapure water, shake and mix, autoclave at 121℃ for 20min, wait until the temperature drops to about 60℃, take it out of the autoclave, place it in a safety cabinet, add 2ml anti-penicillin streptomycin solution, shake and mix, introduce it into the prepared culture dish, roughly pour it to three-quarters of the bottom of the culture dish, shake the culture dish to make the culture medium evenly distributed throughout the bottom of the dish, irradiate the culture dish with ultraviolet light for 30min (while waiting for the culture medium to solidify), add 10ul TM-WT or TM-LUC bacterial solution to the center of the culture medium, wait for the bacterial solution to solidify, seal the culture dish with a seal, put it upside down in a fresh-keeping bag, expel the air, close it and put it in a 27℃ incubator. Take 13g brain heart infusion agar powder, repeat the above steps, and finally put it in a 37℃ incubator.

[0104] (2) Record of colony growth

[0105] From the first day of colony culture, take photos of the front and back of the colonies in the culture dish for 18 consecutive days; measure the colony diameter with the cross method. Take an appropriate amount of colonies from the colonies cultured on days 16, 17, and 18 with sterilized transparent adhesive, place the transparent adhesive on a glass slide (with the bacteria side facing up), use a pipette to drop 1-2 drops of cotton blue stain, cover with a coverslip to allow the stain to be evenly distributed, and observe the morphology under a fluorescence microscope.

[0106] like Figure 4 As shown, Figure 4 Figure 2 shows the growth of TM-WT and TM-LUC, A. the average growth curve (p=0.647) and the average growth rate curve (p=0.912) in PDA medium at 27°C; B. the average growth curve (p=0.619) and the average growth rate curve (p=0.08) in BHI agar medium at 37°C; C. cotton blue staining morphology of TM-WT and TM-LUC on the 16th day, top: TM-WT, bottom: TM-LUC; D. the growth of TM-WT and TM-LUC on PDA and BHI medium on the 14th day, from left to right are WT front, WT back, LUC front, LUC back, top: 27°C, PDA medium, bottom: 37°C, BHI medium.

[0107] The two strains were cultured at 27℃ and 37℃, and the colony growth was recorded for 14 consecutive days. The growth morphology of the two strains in PDA medium was flat and villi-like, and the characteristic red pigment diffusion phenomenon could be observed at the bottom of the colony. After 14 days of culture, the growth diameter of TM-WT was 6.26±0.65cm, and the growth diameter of TM-LUC was 6.45±0.21cm; on BHI medium, it was dark brown and smooth, with a growth diameter of 1.33±0.12cm for TM-WT and 1.43±0.02cm for TM-LUC. After lactic acid phenol cotton blue staining, the two strains were similar in morphology under the microscope, with smooth hyphae and slender branches, and broom-like branches could be found in both strains.

[0108] Example 5

[0109] This example studies the stimulation of THP-1 macrophages by TM-LUC and TM-WT.

[0110] (1) Cell culture

[0111] Cell line: THP-1

[0112] Cell recovery: Take out the THP-1 cell cryovial from -80℃, wrap it with a thin film glove, put it into a 37℃ water bath and shake it back and forth quickly. When the cells are observed to be liquid, take out the cryovial, wipe it with 75% alcohol, put it into a safety cabinet, transfer the cells in the cryovial to 5ml RPMI-1640 culture medium (containing 10% fetal bovine serum, 100U / ml penicillin-streptomycin solution, hereinafter referred to as RPMI-1640 complete culture medium), centrifuge at 900rpm, 22℃ for 5min, discard the culture medium, resuspend the cells with 1ml RPMI-1640 complete culture medium, and then transfer it to a 25T cell culture flask pre-filled with 5ml RPMI-1640 complete culture medium, and put it into a 37℃, 5% CO2 incubator.

[0113] Cell passaging: After 2-3 days of culture, if the color of the culture medium turns yellow, the number of cells observed under the microscope is large, and the cells are aggregated like grape clusters, then cell passaging is performed. Wipe the required equipment and samples with 75% alcohol in advance and put them in a safety cabinet. Expose them to ultraviolet light for 30 minutes. Transfer the cells and culture medium in the 25T culture bottle to a 15ml centrifuge tube with a pipette. Centrifuge at 900rpm and 22℃ for 5 minutes. Discard the culture medium, resuspend the cells with 2ml RPMI-1640 complete culture medium, and transfer 1ml of each to a 75T cell culture bottle pre-filled with 13ml RPMI-1640 complete culture medium, and place it in a 37℃, 5% CO2 incubator.

[0114] Cell seeding plate: If the culture medium of cells turns yellow after about 3-4 days of passage, the cell seeding plate experiment can be carried out. Remove the cells to a 15ml centrifuge tube, centrifuge at 900rpm, 22℃ for 5min, discard the culture medium, resuspend the cells with 1ml RPMI-DMEM complete culture medium, take 10μl of cell suspension, prepare the system 10μl cell suspension + 10μl trypan blue dye + 80μl RPMI-DMEM complete culture medium, mix well and take 10μl to the abalone counting plate for cell counting, take 300,000 cells / well for 12-well plate, take 1 million cells / well for 6-well plate, prepare the sample addition system cell suspension + RPMI-DMEM complete culture medium + PMA (final concentration is 50ng / ml), the sample addition volume per well of 12-well plate is 1ml, and the sample addition volume per well of 6-well plate is 2ml. After the sample addition is completed, mark the well plate and put it into a 37℃, 5% CO2 incubator.

[0115] (2) Co-culture of THP-1 macrophages and bacterial strains

[0116] After 48h of PMA stimulation, THP-1 cells were transformed into THP-1 macrophages. The 12-well culture plate was removed, the upper culture medium was discarded, and PBS was washed three times (500ul for 12-well plate). The infection multiplicity was 1:3, and the system was prepared with RPMI-DMEM complete culture medium + TM-WT and TM-LUC bacterial solution. The sample volume was 1ml per well of the 12-well plate. After the sample was added, the marked well plate was placed in a 37°C, 5% CO2 incubator.

[0117] (3) RNA extraction, reverse transcription and qPCR

[0118] Samples were collected 24 hours and 48 hours after adding TM.

[0119] Extract RNA using Trizol method: Take out the 12-well culture plate, discard the upper culture medium, wash three times with PBS (500ul for 12-well plate), add 250ul Trizol reagent to each well, pipette until the Trizol reagent becomes dilute water, and transfer to a 1.5ml ep tube.

[0120] Add 50ul chloroform reagent (Trizol: chloroform = 5:1) to each ep tube, shake for 15s, mix thoroughly, let stand at room temperature for 3min, centrifuge at 12000g, 4℃ for 15min, pipette 100ul upper colorless aqueous phase into a new ep tube, add 100ul isopropanol reagent (colorless aqueous phase: isopropanol = 1:1) to each tube, shake for 15s, mix thoroughly, let stand at room temperature for 10min, centrifuge at 12000g, 4℃ for 10min, use a pipette to suck out the liquid, leaving the bottom precipitate, add 500ul 75% alcohol to each tube, shake for 2-3s, centrifuge at 12000g, 4℃ for 5min, suck out the alcohol, repeat the alcohol washing operation, discard the alcohol, let stand and wait for the alcohol to evaporate, add 30ul DNA / RNA enzyme-free water to resuspend after evaporation, and incubate in a metal bath at 58℃ for 15min.

[0121] RNA reverse transcription to cDNA: Use PrimerScriptTM RTMaster Mix-RR036A reverse transcription kit from Japan Takara to reverse transcribe RNA samples into cDNA samples. The reverse transcription system is prepared according to the following table (the whole process needs to be performed on ice):

[0122] Table 13 PrimerScriptTM RT Master Mix-RR036A reverse transcription configuration system

[0123]

[0124] Place the 200 μl eight-tube strip in a handheld centrifuge for a split second, then place it in a gradient PCR instrument for reverse transcription reaction according to the following conditions:

[0125] Table 14 PrimerScriptTM RT Master Mix-RR036A reverse transcription conditions

[0126]

[0127] Fluorescence quantitative PCR: GenStar 2×RealStar Green Fast Mixture with ROX Ⅱ kit was used.

[0128] Table 15 GenStar 2×RealStar Green Fast Mixture with ROX Ⅱ Configuration System

[0129]

[0130] Table 16 qPCR reaction condition settings

[0131]

[0132] Example 6

[0133] This example detects the death of THP-1 macrophages using flow cytometry.

[0134] Take out the 6-well culture plate, transfer the upper culture solution to a 15ml centrifuge tube, add 1ml PBS to each well for washing once, put the washing solution into the corresponding centrifuge tube, add 1ml Accutase lysis solution to each well, let it stand at room temperature for 30min, blow up the cells in the well with a pipette, then transfer to the corresponding centrifuge tube, centrifuge at 900rpm, 22℃ for 5min, discard the upper liquid, resuspend the precipitate with 1ml PBS, centrifuge again for 5min under the same conditions, discard PBS, resuspend the precipitate with 300μl, transfer it to a 1.5ml ep tube (blank group, control group, TM-WT group, TM-LUC group), add 2μl PI dye solution to each tube, incubate at room temperature for 5min away from light, and detect on the machine.

[0135] like Figure 5 As shown, Figure 5 Detection of validation factors and cell death in THP-1 macrophages infected with TM-WT and TM-LUC. A. Detection of inflammatory factors in THP-1 macrophages infected with TM-WT or TM-LUC for 24h and 48h. B. Detection of cell death in THP-1 macrophages infected with TM-WT or TM-LUC for 48h and 72h. (* represents p<0.05, ns represents no significant difference).

[0136] TM-WT and TM-LUC were co-cultured with THP-1 macrophages. At 24h and 48h, there was no difference in the expression of TNF-α, IL-1β, and IL-10. At 48h, the killing effect of TM-WT on THP-1 macrophages was about 1.1 times higher than that of TM-LUC, and the difference was statistically significant. At 72h, the killing ability of the two was consistent.

[0137] Example 7

[0138] This example demonstrates TM-LUC luminescence detection and real-time fluorescence imaging of animals.

[0139] like Figure 6 As shown, A. Luminescence detection of luciferin substrate at different time points; B. Detection of TM-LUC fluorescence intensity by imaging instrument.

[0140] In the presence of luciferin substrate, luminescence was observed in TM-LUC tubes but not in TM-WT, demonstrating that TM-LUC can encode LUC.

[0141] The firefly luciferase-labeled Talaromyces marneffei strain constructed by the present invention transfers the firefly luciferase into the TM genome, so that TM can encode luciferase and perform bioluminescence under the action of the substrate luciferin, replacing the traditional ATCC18224 strain. After TM-LUC infects animals, intraperitoneal injection of the luciferin substrate can theoretically make the strain glow, thereby realizing the monitoring of the strain distribution in the animal body; it can also monitor the treatment process after drug administration; and it provides a new direction for the exploration of the mechanism of latent infection.

[0142] The above is only the preferred embodiment of the present application, which does not limit the protection scope of the present application. For those skilled in the art, the present application may have various changes and modifications. Within the spirit and principle of the present application, the changes, modifications, replacements, integrations and parameter changes of these embodiments by conventional substitutions or capable of achieving the same functions without departing from the principles and spirit of the present application shall fall within the protection scope of the present application. Sequence Listing <110> Guangxi Medical University <120> Firefly luciferase-labeled Talaromyces marneffei strain and construction method <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 41 <212> DNA / RNA <213> MICROORGANISM <400> 1 cttgagcaga catcaccatg gaagacgcca aaaacataaa g 41 <210> 2 <211> 37 <212> DNA / RNA <213> MICROORGANISM <400> 2 gaaattcgag ctggtcacct tacacggcga tctttcc 37

Claims

1. A firefly luciferase-labeled Talaromyces marneffei strain, named TM-LUC strain, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and its microbiological accession number is: CGMCC No.40152, deposited on March 25, 2022; the construction method of the firefly luciferase-labeled Talaromyces marneffei strain comprises the following steps: Step S1, constructing a firefly luciferase recombinant plasmid, specifically comprising: S11, luciferase fragment amplification, using pGL3-basic plasmid as template, designing primers and amplifying luciferase fragment; the primer sequences include: The luciferase F primer sequence was 5′-cttgagcagacatcaccatggaagacgccaaaaacataaag-3′; The luciferase R primer sequence was 5′-gaaattcgagctggtcaccttacacggcgatctttcc-3′; S12, Ncol, BstEI digested the pCAMBIA1303-TrpC-Hygro-gpdA-GFP vector backbone; S13, recombination connection, the recovered PCR product and the pCAMBIA1303-TrpC-Hygro-gpdA-GFP vector backbone recovered after enzyme digestion were recombined and connected, reacted at 37°C for 30 minutes, and then quickly placed on ice for cooling; then the recombinant product was transformed, TranT1 competent cells were thawed on ice, 10ul of the recombinant product was added to 100ul of competent cells, the tube wall was gently tapped to mix, and it was allowed to stand on ice for 30min; after heat shock in a 42°C water bath for 45s, it was immediately placed on ice for cooling for 2-3min, LB medium was added, and the bacteria were shaken at 37°C for 1h; the LB solid culture medium plate with corresponding resistance was preheated in a 37°C incubator, centrifuged at 5,000rpm for 5min, and 900ul of supernatant was discarded; the bacteria were resuspended with the remaining culture medium, and gently spread on a plate containing 50ug / ml kanamycin with a sterile coating, and inverted incubator at 37°C for 12-16h; S14, recombination product identification, picking up more than 100 clones on the recombination reaction transformation plate for colony PCR identification; step S2, transfer to TM genome, specifically including: S21. Agrobacterium transformation culture, including:

210. Take EHA105 competent cells, add plasmid pCAMBIA1303-TpC-Hygro-gpdA-Luc, and mix well; 211. Add LB liquid medium without antibiotics and culture at 28°C with shaking for 2-3 hours; 212. Collect the bacteria by centrifugation, keep the supernatant, gently blow and resuspend the bacteria, and spread it on the LB plate with the corresponding antibiotics. Invert it and place it in a 28℃ incubator for 2-3 days. Pick the positive clones and transfer them to LB medium and shake at 28℃. S22, Agrobacterium infection with Talaromyces marneffei, including:

221. Add 1% of EHA105 bacterial solution containing plasmid pCAMBIA1303-TrpC-Hygto-gpdA-Luc into TM and culture with shaking at 28°C for 24 h.

222. Add glass beads to the bacterial solution after co-culture, shake to break up the clumped bacteria, then spread on a solid Sabouraud glucose medium plate containing hygromycin, and culture in a 37°C incubator until a single clone grows out; 223. The monoclonal transformant was transferred to a new liquid Sabouraud medium containing hygromycin, cultured in a 37°C incubator with shaking until the logarithmic growth phase, and the genome was extracted for identification.

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