Construction method and application of random mutant saturated library of non-essential genes of Mycobacterium bovis

Abstract

The invention relates to the technical field of biology, and particularly discloses a construction method and application of a mycobacterium bovis non-essential gene random mutant saturated library. The construction method of the mycobacterium bovis non-essential gene random mutant saturated library comprises the following steps: infecting mycobacterium bovis C68001 with a phage MycoMar T7, and constructing a transposon mutant library; wherein the bacteriophage MycoMar T7 carries a Himarl transposon, and the bacteriophage MycoMar T7 can be used for preparing a recombinant According to the method, the saturated library with the storage capacity of 27648 and covering 99.9% of non-essential gene mutants of the Mycobacterium bovis is constructed, and a good foundation is laid for researching gene functions and pathogenic mechanisms of the Mycobacterium bovis and developing drugs and vaccines.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis and an application thereof. Background Art

[0002] Mycobacterium tuberculosis variant bovis (M. tuberculosis variant bovis) is a member of the Mycobacterium tuberculosis complex (MTBC), which can infect a variety of wild animals and livestock, and can also infect humans through contact with sick animals or ingestion of infected animal products. According to statistics from the World Health Organization, there were 121,268 cases of tuberculosis worldwide in 2010, of which 10,545 deaths were related to infection with M. tuberculosis variant bovis. In-depth research on M. tuberculosis variant bovis is crucial to understanding the evolution of tuberculosis pathogens, host adaptability, and pathogenic mechanisms, and also provides a theoretical basis for the development of new tuberculosis vaccines and diagnostic tools.

[0003] In the field of phenotypic screening and regulatory network analysis, mutant libraries play a vital role. As an efficient library construction method, transposon mutagenesis technology can quickly generate a large number of mutants, and each mutation site can be precisely located by the inserted transposon sequence.

[0004] Mycobacterium tuberculosis bovis is a slow-growing bacterium. It takes 10 to 14 days to culture in liquid medium, 3 to 4 weeks to culture in solid medium, and 5 to 8 months to construct a gene-deficient strain. The time cost of functional gene and phenotypic research is enormous. However, there is currently no report on a mutant library of Mycobacterium tuberculosis bovis internationally. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis. The constructed library has a large capacity and is conducive to subsequent applications.

[0006] In order to solve the above technical problems, the present invention provides a method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis, characterized in that it comprises the steps of infecting Mycobacterium bovis C68001 with bacteriophage MycoMar T7 to construct a transposon mutation library;

[0007] Wherein, the bacteriophage MycoMar T7 carries the Himarl transposon.

[0008] As an improvement of the above technical solution, in the step of infecting bovine Mycobacterium C68001 with bacteriophage MycoMar T7 to construct a transposon mutation library, the titer of the bacteriophage MycoMar T7 is ≥3×10 11 PFU / mL; and / or

[0009] The infection conditions include: MOI=8-12, temperature of 35°C-40°C, and time of 2h-5h.

[0010] As an improvement of the above technical solution, in the step of infecting bovine Mycobacterium C68001 with bacteriophage MycoMar T7 to construct a transposon mutation library, the titer of the bacteriophage MycoMar T7 is 5×10 11 PFU / mL; and / or

[0011] The infection conditions included: MOI=10, temperature of 37°C, and time of 3h.

[0012] As an improvement of the above technical solution, in the step of infecting Mycobacterium bovis C68001 with bacteriophage MycoMar T7 to construct a transposon mutation library, the infection is followed by dilution with MP buffer at a multiple of 15 to 30 times.

[0013] As an improvement of the above technical solution, in the step of infecting Mycobacterium bovis C68001 with bacteriophage MycoMar T7 to construct a transposon mutation library, MP buffer is used to dilute after infection, and the dilution multiple is 20 times.

[0014] As an improvement of the above technical solution, the method further includes screening the bacteriophage MycoMar T7 to obtain the temperature-sensitive bacteriophage MycoMar T7.

[0015] As an improvement of the above technical solution, the bacteriophage MycoMar T7 was inoculated into the culture medium containing Mycobacterium smegmatis M. 2 155 bacterial solution and top agar plates were used to screen for phages that could proliferate at 30°C but not at 37°C, which was the temperature-sensitive phage MycoMar T7.

[0016] As an improvement of the above technical solution, the method further includes the step of culturing and amplifying the temperature-sensitive bacteriophage MycoMar T7;

[0017] Among them, Mycobacterium smegmatis M.Smegmatis mc 2 155% of the bacterial solution was used to culture and amplify MycoMar T7.

[0018] Accordingly, the present invention also discloses the use of the above-mentioned method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis in any one of (1) to (5):

[0019] (1) preparing a kit for detecting Mycobacterium bovis;

[0020] (2) preparing a Mycobacterium bovis vaccine;

[0021] (3) Screening of Mycobacterium bovis vaccines;

[0022] (4) preparing a medicament for treating Mycobacterium bovis;

[0023] (5) Screening drugs for treating bovine Mycobacterium.

[0024] The implementation of the present invention has the following beneficial effects:

[0025] In the method for constructing a saturated library of random mutants of non-essential genes of Mycobacterium bovis in one embodiment of the present invention, bacteriophage MycoMar T7 is used to infect Mycobacterium bovis C68001 to construct a transposon mutation library. Mycobacterium bovis C680001 has the advantages of strong pathogenicity and low spontaneous mutation rate. By infecting it with a specific bacteriophage, a high-capacity transposon mutation library can be successfully obtained. Specifically, the mutant library constructed by the present invention has a capacity of 27648, covering 99.9% of non-essential genes of Mycobacterium bovis, providing a technical platform for studying the gene function, pathogenic mechanism and vaccine development of Mycobacterium bovis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of the evaluation of the pathogenicity of Mycobacterium tuberculosis bovine variants C68001 and C68002. A: Mouse weight; B: Liver index; C: Spleen index; D: Lung index; E: Organ bacterial load.

[0027] Figure 2 This is the complete genome sequence of the bovine variant of Mycobacterium tuberculosis C68001-P1.

[0028] Figure 3 The results of the bacteriophage temperature sensitivity test are shown in Figure 1. A: Incubated at 30°C, plaques are visible; B: Incubated at 37°C, no plaques are seen.

[0029] Figure 4 This is the gel electrophoresis diagram of the identification of mutant single colony PCR products. Lane M: DNA 2000 Marker; Lane +: phage DNA; Lanes 2-7: 7 randomly selected mutants; Lane -: C68001 genome.

[0030] Figure 5The scanning electron micrographs of the Rv3671c gene mutant strains are shown in Figure 1. A: the parent strain C68001-P1; B: the strain overexpressing the Rv3671c gene; C: the strain complementing the Rv3671c gene; D: the strain lacking the Rv3671c gene. DETAILED DESCRIPTION

[0031] For ease of understanding of the present invention, the present invention will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific implementation or embodiment, and are not intended to limit the present invention. The optional scope of the term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the combinations include any two related listed items, any more related listed items, or all related listed items.

[0033] The following examples are given for the purpose of illustrating various embodiments of the present invention and are not intended to limit the present invention in any way.

[0034] The present invention. Those skilled in the art will understand that the changes and other uses thereof are within the spirit of the present invention as defined by the scope of the claims. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. The promoter and terminator sequences mentioned in the examples can also be downloaded from NCBI, and the specific sequence start positions can be obtained based on the primers in the primer table. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0035] In the present invention, "first aspect", "second aspect" and the like are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features.

[0036] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0037] The percentage content involved in the present invention, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0038] The percentage concentrations involved in the present invention, unless otherwise specified, refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0039] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0040] Example 1 Screening and identification of parent strains

[0041] 1. Pathogenicity detection of Mycobacterium tuberculosis bovine variant C68001, C68002, and C68001-5. Frozen Mycobacterium tuberculosis bovine variant C68001 (CVCC68001, National Veterinary Microbiological Bacteria (Virus) Collection Center) and C68002 (CVCC68002, National Veterinary Microbiological Bacteria (Virus) Collection Center) were streaked on plates, and single bacteria were picked and inoculated into 7H9-OADC-Ty medium for 14 days. When the strains grew to the logarithmic phase (OD 600 =0.8-1.0), 10 mL of bacterial solution was added with an equal volume of 50% glycerol, and the solution was divided and frozen in a -80°C refrigerator to be used as seed markers C68001-P1 and C68002-P1. The remaining bacterial solution was centrifuged to collect the cells, washed three times with sterile PBS, and ultrasonically dispersed for 1 min before diluting the solution to OD600 = 1.0. At this time, the number of viable cells was 3 × 10 8 CFU / mL, diluted to 5 × 10 5 CFU / mL. Twenty 6-week-old C57BL / 6J mice were randomly divided into three groups, with 6 mice in each group. After intramuscular injection of Zotai 50 for anesthesia, each group was intranasally dripped with PBS and diluted bacterial solution, 20 μL / mouse. After continuous feeding for 4 weeks, the mice were dissected, and the weight, liver, spleen, and lung weights were weighed. The organ coefficient ratio was calculated, and the tissue was ground and graded diluted, and then coated on 7H10-OADC plates to calculate the organ bacterial load (see the results). Figure 1 ), the results showed that C68001-P1 was significantly more pathogenic to mice than C68002-P1, therefore, C68001 was preferred as the mother strain.

[0042] 2. Full genome sequence determination

[0043] The frozen C68001-P1 strain was inoculated into 7H9-OADC-Ty medium, and the genome was extracted. Based on Nanopore third-generation and second-generation sequencing technologies, the complete genome map of the bacteria was drawn. The complete genome size of C68001 was 4.342M, containing 3998 genes, 73389 TA' sites, and a GC content of 65.64% ( Figure 2 ). The drawing of the whole genome map provides a reference for locating the transposon insertion site.

[0044] Example 2 Preparation of MycoMar T7 phage

[0045] 1. Amplification of temperature-sensitive phage

[0046] Take frozen Mycobacterium smegmatis M.Smegmatis mc 2 155 (see CN112746051A) was streaked onto a 7H10-OADC plate, and a single colony was picked and transferred to 10 mL of 7H9-OADC-Ty medium. The culture was shaken at 37°C and 110 rpm until the plateau phase (OD 600 =2.0), and then transferred to 7H9-OADC-Ty medium at a volume ratio of 1:100, and cultured with shaking until OD 600 =1.0. The ΦMycoMarT7 virus strain was diluted 10 times in a series with MP buffer, and 100 μL of the diluted MycoMar T7 phage (Isolation, Identification and Functional Study of Pathogenic Genes of Mycobacterium marinum, Yu Jia, 2010, Fudan University) was mixed with 300 μL of bacterial solution, incubated at 37°C for 30 min, added to 6 mL of pre-melted Top Agar, inverted to mix, and immediately poured into a 7H10 plate, cooled and solidified at room temperature, and then inverted and cultured at 30°C for 3 days.

[0047] 2. Screening and titer determination of temperature-sensitive phages

[0048] Add 500 μL of OD 600= 1.0 of Mycobacterium smegmatis liquid, add 6mL of pre-melted Top Agar, mix by inversion, and immediately pour into 7H10 plate, cool and solidify at room temperature. Randomly pick 50 plaques from the plate amplified with phages and inoculate them synchronously on 2 plates with Mycobacterium smegmatis, and culture them at 30℃ and 37℃ for 3d, respectively. Pick 10 plaques that proliferate at 30℃ but not replicate at 37℃, add them to EP tubes, add 200μL MP buffer, incubate at 4℃ overnight, centrifuge and take supernatant, perform continuous 10-fold gradient dilution with MP buffer, take 100μL of diluted phage and mix with 300μL of bacterial liquid, incubate at 37℃ for 30min, add Top Agar, mix by inversion, and immediately pour into 7H10 plate, cool and solidify at room temperature, and then invert and culture at 30℃ for 3d. Select a plate with closely connected plaques, add 2 mL of MP buffer, scrape the agar, rotate at 4°C overnight, centrifuge, take the supernatant, and filter through a 0.22 μm filter for sterilization.

[0049] Take the selected temperature-sensitive phage and continuously amplify it for 3 generations. Dilute the 3rd generation phage 10 times with MP buffer. Take 100 μL of the diluted phage and mix it with 300 μL of bacterial solution. Incubate at 37℃ for 30 minutes, add Top Agar, mix it upside down and pour it into 7H10 plate immediately. After cooling and solidifying at room temperature, turn it upside down and culture it at 30℃ for 3 days. Select plates with 30-300 plaques for counting and calculating the phage titer. And randomly select several plaques to retest the temperature sensitivity.

[0050] After screening different phage clones, we finally identified a phage with stable temperature-sensitive properties and a titer of 5×10 11 PFU / mL phage was used for library construction.

[0051] Example 3 Optimization of infection conditions

[0052] 1. Determination of the optimal infection ratio

[0053] Take C68001-P1 and streak it on a 7H10-OADC plate, pick a single colony and transfer it to 10mL 7H9-OADC-Ty medium until the stationary phase, inoculate it into 50mL 7H9-OADC-Ty at a volume ratio of 1:100 and culture it for 2 weeks until OD600 = 0.8-1.0. After centrifugation, remove the supernatant, add MP buffer to wash 3 times, and resuspend in 5mL MP buffer. 1mL of bacterial solution is spread on a 7H10-OADC-kana plate to detect the spontaneous mutation rate. 100μL of bacterial solution is diluted 10 times continuously to calculate CFU. 3 mL of bacterial solution was infected at MOI (bacteriophage: bacteria) = 10, 5, and 1 at 37°C for 24 h, coated on 7H10-OADC-kana plates, and cultured continuously at 37°C for 3 to 4 weeks. The positive clones were approximately 2000, 1000, and 70, respectively. Therefore, MOI = 10 is the best infection ratio.

[0054] 2. Optimization of optimal infection time and bacterial dilution concentration

[0055] Since there were too many positive clones obtained at 24 hours of infection, it was difficult to obtain a single clone. Therefore, at an infection ratio of MOI = 10, the reaction was carried out at 37°C for 3 hours and 24 hours respectively, and the bacterial solution after the reaction was diluted with MP buffer to 2 times, 20 times, 100 times and 200 times for plating. After continuous culture for 3 to 4 weeks, there were still too many clones obtained at 24 hours of infection, and the condition of reacting for 3 hours and plating with 20 times dilution was most suitable for picking single colonies.

[0056] Example 4 Establishment of transposon mutation library

[0057] Amplify 100mL of C68001-P1, wash and resuspend in 10mL MP buffer. Take 5mL of bacterial solution and add phage at MOI=10, shake and mix, and incubate at 37℃ for 3h. The infected bacterial solution is spread on 7H10-OADC-kana plates at a 20-fold dilution and cultured at 37℃ for 3-4 weeks. Take the remaining 1mL of concentrated bacterial solution and spread it on 7H10-OADC-kana plates to detect the spontaneous mutation rate. Take another 100μL of bacterial solution and perform 10-fold continuous gradient dilution, spread it on 7H10-OADC plates to determine CFU. When a single colony grows on the plate, pick a single colony and inoculate it into 288 7H9-OADC-Ty-kana 96-well cell culture plates pre-added with 200 μL / well, culture at 37°C for 2-3 weeks, add glycerol with a final concentration of 20%, and freeze at -80°C. A total of 27,648 transposon random mutation strains were obtained. The count result of the concentrated bacterial solution used for this library construction was 3×10 9 CFU / mL, transduction efficiency was 3.38×10 -6 , the spontaneous mutation rate is 6.67×10 -10Seven single colonies were randomly selected and PCR amplified for the kanamycin resistance gene aphⅡ. The band size and sequencing results were correct, confirming that the growing clones were all transposon mutants ( Figure 5 ). C68001-P1 has 4093 genes, including 73389 TA' sites, 3164 non-essential genes, about 53214 of which are located on non-essential genes, and the median number of TA' sites in non-essential genes is 12. It is estimated that there are 4435 (53214 / 12) non-essential genomic regions. The library capacity required for each non-essential gene to be inserted at least once is calculated. The relationship between the library capacity E(T) and the number of non-essential genes n can be expressed as:

[0058]

[0059] Based on this calculation, 27648 mutant strains can cover 99.9% of the non-essential genes in the genome of Mycobacterium tuberculosis bovine variant C68001.

[0060] Example 5 Application of transposon mutation library

[0061] A plate of 96 strains from the library was randomly selected, and the whole plate of strains was inoculated into 7H9-OADC and Sutong medium at a ratio of 1:100, and cultured at 37℃ for 2-3 weeks. The biofilm formation was observed with C68001-P1 as a reference. Among them, 6 biofilms had obvious defective phenotypic mutations. After they were expanded, the bacterial genome was extracted, and after BamHI digestion and T4 Ligase ligation, the ligation product was transferred into Escherichia coli competent pir-116. Positive colonies were picked on LB (containing 50μg / ml kanamycin) plates, and plasmids were extracted after liquid expansion. The flanking fragment sequence of the transposon insertion region was identified by DNA sequencing. With the C68001 genome sequence as a reference, the insertion sites were determined to be Rv1980c, Rv1096, Rv3425, Rv3136, Rv2589 and Rv3671c. Scanning electron microscopy revealed that after the deletion of the Rv3671c gene, the bacterial surface was smooth and had no hyphae, further confirming that the saturation library of random mutants of non-essential genes of the bovine variant of Mycobacterium tuberculosis constructed in this study can be used for high-throughput rapid screening of the bovine variant of Mycobacterium tuberculosis, and is an efficient tool for studying the gene function, pathogenic mechanism, and development of drugs and vaccines of the bovine variant of Mycobacterium tuberculosis.

[0062] The technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical scheme of the present invention in a specific and detailed manner, but it cannot be understood as a limitation on the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the scope of protection of the present invention.

[0063] It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the attached claims of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis, characterized in that: The method comprises the steps of infecting bovine Mycobacterium C68001 with bacteriophage MycoMar T7 to construct a transposon mutation library; Wherein, the bacteriophage MycoMar T7 carries the Himarl transposon.

2. The method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis according to claim 1, characterized in that: In the step of infecting bovine Mycobacterium C68001 with bacteriophage MycoMar T7 to construct a transposon mutation library, the titer of the bacteriophage MycoMar T7 is ≥ 3×10 11 PFU / mL; and / or The infection conditions include: MOI=8-12, temperature of 35°C-40°C, and time of 2h-5h.

3. The method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis according to claim 1, characterized in that: In the step of infecting bovine Mycobacterium C68001 with bacteriophage MycoMar T7 to construct a transposon mutation library, the titer of the bacteriophage MycoMar T7 is 5×10 11 PFU / mL; and / or The infection conditions included: MOI=10, temperature of 37°C, and time of 3h.

4. The method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis according to claim 1, characterized in that: In the step of infecting bovine Mycobacterium C68001 with bacteriophage MycoMar T7 to construct a transposon mutation library, the infection is followed by dilution with MP buffer at a dilution multiple of 15 to 30 times.

5. The method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis according to claim 1, characterized in that: In the step of infecting bovine Mycobacterium C68001 with bacteriophage MycoMar T7 to construct a transposon mutation library, the infection was followed by dilution with MP buffer at a dilution multiple of 20 times.

6. The method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis according to claim 1 or 6, further comprising the step of screening bacteriophage MycoMar T7 to obtain temperature-sensitive bacteriophage MycoMar T7.

7. The method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis according to claim 6, characterized in that: The bacteriophage MycoMar T7 was inoculated into the culture medium containing Mycobacterium smegmatis M. 2 155 bacterial solution and topagar plates were used to screen for phages that could proliferate at 30°C but not at 37°C, which was the temperature-sensitive phage MycoMar T7.

8. The method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis according to claim 6, characterized in that: The method also includes the step of culturing and amplifying the temperature-sensitive bacteriophage MycoMar T7; Among them, Mycobacterium smegmatis M.Smegmatis mc 2 155% of the bacterial solution was used to culture and amplify MycoMar T7.

9. Use of the method for constructing a saturation library of random mutants of non-essential genes of Mycobacterium bovis according to any one of claims 1 to 8 in any one of (1) to (5): (1) preparing a kit for detecting Mycobacterium bovis; (2) preparing a Mycobacterium bovis vaccine; (3) Screening of Mycobacterium bovis vaccines; (4) preparing a medicament for treating Mycobacterium bovis; (5) Screening drugs for treating bovine Mycobacterium.

Images