CN109762791B

CN109762791B - Recombinant adenovirus containing BPI-Fc chimeric gene and application thereof

Info

Publication number: CN109762791B
Application number: CN201811374590.9A
Authority: CN
Inventors: 陈金栋; 安云庆; 李劲超; 杨密清
Original assignee: Anjun Beijing Gene Technology Co ltd; Xiamen Lianhe Anjin Biological Engineering Co ltd
Current assignee: Anjun Beijing Gene Technology Co ltd; Xiamen Lianhe Anjin Biological Engineering Co ltd
Priority date: 2018-11-19
Filing date: 2018-11-19
Publication date: 2021-02-09
Anticipated expiration: 2038-11-19
Also published as: WO2020103412A8; CN109762791A; WO2020103412A1

Abstract

The present invention relates to the field of gene therapy. In particular, the invention relates to a recombinant adenovirus for treating drug-resistant gram-negative bacterial infection, and preparation and application thereof, wherein the recombinant adenovirus comprises a BPI-Fc chimeric gene of a fusion protein for coding a Bactericidal/penetration enhancing protein (BPI) and an immunoglobulin heavy chain constant region Fc. The invention also relates to the use of said recombinant adenovirus for the preparation of a pharmaceutical composition for the treatment of drug-resistant gram-negative bacterial infections.

Description

Recombinant adenovirus containing BPI-Fc chimeric gene and application thereof

Technical Field

Background

Antibiotic RESISTANCE poses a significant threat to public health and human health, WHO has called a call in recent years and caused worldwide importance to cope with Global RESISTANCE infection, alerting humans to enter the post-antibiotic age [ antibiotic RESISTANCE scientific public Report on successful human 2014, WHO ]. WHO quotes from Jim O' Neill in the united kingdom [ antibiotic Resistance on the Global agenda. dec 1,2015], estimated that about 70 million people die from drug-resistant bacterial infections every year worldwide, and that 1000 million people would be expected to die from drug-resistant bacterial infections every year by 2050 if no effective measures were taken. The bacterial multi-drug resistance and super-drug resistance problems are highlighted, wherein Gram-negative bacteria (GNB), one of the main pathogens causing human infectious diseases, has strong resistance to clinically common antibiotics and is the drug-resistant bacteria which are currently most concerned [ https:// en. In 2017, the WHO firstly publishes a list of 12 drug-resistant bacteria which form the greatest threat to human health, wherein 9 of the bacteria are gram-negative bacteria [ Nature 543,15(02 March 2017) doi: 10.1038/nature.2017.21550 ] [ https:// news.un.org/zh/store/2017/02/271472 ]. In particular, it is pointed out that: plasmid-mediated super carbapenem resistance gene NDM-1 was found in Klebsiella pneumoniae in 2009 and plasmid-mediated super colistin resistance gene mcr-1 was found in Escherichia coli in 2015 [ Antimicrob Agents Chemother.2009; 53(12) (5046) (Lancet Infect Dis.2010; 597] [ Lancet Infect Dis.2016; 16(2) 161] to almost completely prevent the last line of defense of antibiotics for clinical treatment of gram-negative bacterial infections. With the increasingly serious antibiotic resistance and the coming post-antibiotic age, the research and development of antibiotics for replacing biological medicines has urgency and great application prospect.

Disclosure of Invention

In one aspect of the invention, a recombinant adenovirus comprising a chimeric gene encoding a BPI-Fc fusion protein, wherein said chimeric gene comprises a coding sequence for a functional fragment of human BPI and a human immunoglobulin heavy chain constant region Fc gene, wherein the 3' end of the coding sequence for the functional fragment of human BPI is linked to the Fc gene directly or through a human immunoglobulin hinge region.

In one embodiment, the adenovirus of the invention is an adenovirus vector selected from the group consisting of serotypes: ad5, Ad2, and Ad55 serotypes; preferably of the Ad5 serotype.

In a preferred embodiment, the functional fragment of human BPI according to the invention is selected from human BPI_1-199Fragments and human BPI_1-193Fragments, preferably human BPI_1-199And (3) fragment.

In one embodiment, the human immunoglobulin heavy chain constant region Fc gene of the present invention is selected from the group consisting of C γ 1, C γ 2, C γ 3, C α 1, C α 2 and C μ genes or alleles thereof, preferably the C γ 1 gene.

In another embodiment of the invention, the chimeric gene encoding a BPI-Fc fusion protein has, in order from 5 'to 3': human BPI signal peptide coding sequence, BPI_1-199The coding sequence, the human immunoglobulin hinge region and the coding sequence for Fc γ 1 are preferably linked at the 5 'and 3' ends, respectively, with the CMV promoter and SV40poly a expression control elements. In one embodiment, the nucleotide sequence of the BPI-Fc γ 1 chimeric gene encoding the BPI-Fc γ 1 fusion protein is as set forth in SEQ ID NO: 1, preferably wherein BPI_1-199The coding sequence of amino acid residues 4 to 24 of (a) is modified to SEQ ID NO: 2.

in a preferred embodiment, the present invention provides Ad5-BPI-Fc γ 1 recombinant adenovirus capable of infecting animal cells and expressing BPI-Fc γ 1 fusion protein for direct killing and rapid and efficient killing of multi-drug resistant gram negative bacteria by complement activation and opsonophagocytosis, and which has been successful in the treatment of animals in a model of multi-drug resistant gram negative bacteria infection in vivo.

In yet another aspect, the invention provides the use of the recombinant adenovirus for the treatment of drug-resistant gram-negative bacterial infections, wherein the drug-resistant gram-negative bacteria comprise multi-drug-resistant gram-negative bacteria, in particular drug-resistant gram-negative bacteria with a multi-drug-resistant mechanism. Specifically, the drug-resistant gram-negative bacteria include escherichia coli, acinetobacter baumannii, and klebsiella pneumoniae. In particular aspects, the drug-resistant gram-negative bacterium carries a drug-resistant gene that is a super-broad-spectrum drug-resistant gene ESBLs or a super-drug-resistant gene NDM-1.

In another aspect, the invention provides a pharmaceutical composition for treating a drug-resistant gram-negative bacterial infection, comprising a therapeutically effective amount of a recombinant adenovirus of the invention, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a use of the recombinant adenovirus in the preparation of a medicament for treating drug-resistant gram-negative bacteria infection, wherein the drug-resistant gram-negative bacteria comprise multiple drug-resistant gram-negative bacteria, and particularly relates to drug-resistant gram-negative bacteria with multiple drug-resistant mechanisms. Specifically, the drug-resistant gram-negative bacteria include escherichia coli, acinetobacter baumannii and klebsiella pneumoniae. In particular aspects, the drug-resistant gram-negative bacteria carry a drug-resistant gene that is an extended spectrum drug-resistant gene ESBLs or an extended drug-resistant gene NDM-1.

In another aspect of the present invention, there is provided a method for preparing a recombinant adenovirus comprising a nucleotide sequence encoding a fusion protein of BPI and Fc, the method comprising the steps of:

(a) providing an adenovirus backbone vector;

(b) providing a shuttle expression vector carrying an expression cassette for the BPI and Fc fusion protein coding sequences;

(c) co-transfecting the adenovirus backbone vector of step (a) and the shuttle expression vector of step (b) into a host cell for homologous recombination to obtain a recombinant adenovirus.

In a preferred embodiment, the method further comprises the steps of:

(d) infecting host cells with the recombinant adenovirus obtained in step (c), and

(e) the recombinant adenovirus is obtained from the infected host cell.

Detailed Description

Bactericidal/penetration enhancing protein (BPI) is a cationic antimicrobial protein with a molecular weight of about 55KD first found in human polymorphonuclear neutrophils by Weiss et al in 1978, consisting of 456 amino acid residues; n-terminal functional fragment (N-terminal) thereofBPI of amino acid residues 1-199 of the region_1-199Fragments and their C-terminal 6 amino acid residues truncated 1-193 BPI_1-193Fragment) has the same high affinity binding to Lipopolysaccharide (LPS) and lipid a of gram-negative bacteria as human native BPI, Endotoxin (Endotoxin) neutralization, and direct killing of gram-negative bacteria. XOMA corporation developed recombinant human BPI N-terminal functional fragments starting from 1990 s: (

rBPI₂₁) And a plurality of clinical trials are carried out, but the sterilization of BPI needs to last for a long time (>3 hours) maintain a higher concentration (>10nM), and rBPI₂₁Short half-life in vivo, large therapeutic dose, difficulty in maintaining effective therapeutic concentrations in vivo, and the like, and has not been clinically successful and approved by the FDA. [ J.biol.chem.253:2664(1978)][J Biol Chem.264:9505(1989)][J.Exp. Med.174:649(1991)][J Clin Invest.90:1122(1992)][Cazzola,et al. Curr Opin Pulm Med,10:204(2004)][Mannion,et al.J.Clin.Investig. 85:853(1990)][Shock.10:161(1998)][J.Trauma.46:667(1999)] [Lancet.356:961(2000)][Crit Care Med 2001 29(7)(Suppl.):S130-S135] [ASM News.68:543(2002)]

Immunoglobulins (igs) comprise a family of proteins with many similar structures but differing in important structure leading to different antigen binding properties and other biological activities. Human igs can be divided into five classes, IgG, IgA, IgM, IgD and IgE, of which three classes, IgG, IgA and IgM, also have subclasses and play an important role in the immune defense against infection. Heavy chain constant region Fc of IgG (Fragment crystalline, Fc; the coding sequence of Fc is called Fc gene): c gamma 1, C gamma 2 and C gamma 3 have double functions of activating complement and mediating opsonophagocytosis; heavy chain constant region Fc of IgA: c alpha 1 and C alpha 2 have the function of mediating, regulating and phagocytizing; heavy chain constant region Fc of IgM: mu has a powerful complement activating function and can bind to macrophages via C1b to promote phagocytosis (although not independently opsonize phagocytosis). Medical Immunology ISBN: 9787117208215

The Ig-like BPI-Fc fusion protein formed by the BPI or the N-terminal functional fragment thereof and the Ig heavy chain constant region Fc or the equivalent body thereof in a chimeric way can have the dual functions of BPI and immunoglobulin Fc, namely has BPI targeting combination with LPS and lipoid A and direct killing of gram-negative bacteria, also has the functions of activating complement through Fc and opsonophagocytizing to rapidly kill the gram-negative bacteria, and has the action mechanism capable of overcoming the drug resistance of gram-negative bacteria.

Gene therapy (Gene therapy) is a method of treating diseases by introducing functional genes into organs, tissues or cells in vivo and expressing them. Gene therapy vectors fall into two broad categories: viral vectors (mainly including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors and lentivirus vectors, and in addition, vaccinia virus vectors, poxvirus vectors, herpes simplex virus vectors and the like are used in a small amount) and non-viral vectors (mainly including naked DNA, liposomes, nano-vectors and the like). The adenovirus vector has the characteristics of wide host cell range, capability of effectively infecting divided cells and non-divided cells, DNA (deoxyribonucleic acid) which is not integrated with host cell genomes, no insertion mutation risk, strong immunogenicity, difficulty in repeated treatment and the like, and the target gene is continuously expressed in vivo for 2-3 weeks. [ Anderson, Nature,392:25(1998) ] [ Francis S.Collins, Scott Gottlieb.the Next Phase of Human Gene-Therapy overhead.2018 (https: i.org/10.1056/NEJMP1810628) ].

PCT/CN2005/000986, CN 200580000538.1 disclose a recombinant adeno-associated virus containing BPI-Fc chimeric gene, which is used for animal treatment of gram-negative bacteria [ E.coli O111: B4(CMCC (B) No. 44101-9; antibiotic sensitivity ] infection model in mice, in view of the fact that clinically severe gram-negative bacterial infection develops into severe drug resistance to antibiotics and needs to be treated effectively in medium and short term, the inventor further provides the technical scheme of the invention through intensive research, constructs a recombinant adenovirus containing BPI-Fc chimeric gene, proves that the recombinant adenovirus succeeds in the treatment of multiple drug resistance (carrying drug resistance gene) gram-negative bacteria in-vitro experimental model and in-vivo infection model animals, and at the same time, firstly, the adenovirus vector is used for mediating the target gene not to be integrated with host cell genome, The sustained and high-efficiency expression in vivo is realized for 2-3 weeks, and the antibiotic has remarkable advantages and practicability when used as a drug-resistant gram-negative bacteria infection medium-short term antibiotic substitution treatment drug.

Further objects and implementations of the invention will be set forth below, and the scope, content and advantages of the invention will be apparent to those skilled in the art. While the invention provides the preferred embodiments, those skilled in the art will recognize that various modifications and changes are also within the scope of the present description.

The gene therapy viral vector mainly comprises an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector and the like. The adenovirus vector mainly comprises common serotypes such as Ad5, Ad2 and Ad55, has wide host cell range, can effectively infect dividing cells and non-dividing cells, DNA is not integrated with host cell genomes, the risk of insertion mutation does not exist, and the target gene is continuously expressed in vivo for 2-3 weeks. In view of the fact that clinically severe gram-negative bacterial infection develops into severe antibiotic resistance, the effective intermediate-short term antibiotic substitution treatment drug has great advantages and application prospects. The invention constructs a recombinant adenovirus containing BPI-Fc chimeric gene as a medium-short term antibiotic substitution treatment drug for drug-resistant gram-negative bacteria infection.

BPI N-terminal functional fragment BPI_1-199And BPI with 6 amino acids truncated at C terminal_1-193Has the same endotoxin neutralizing and gram-negative bacteria killing effects as natural human BPI, preferably BPI_1-199。

IgG Fc: c gamma 1, C gamma 2 and C gamma 3 have double functions of activating complement and mediating opsonophagocytosis; IgA Fc: c alpha 1 and C alpha 2 have the function of mediating, regulating and phagocytizing; IgM Fc: mu has a powerful complement activating function and can bind to macrophages via C1b to promote phagocytosis (but not independently opsonize phagocytosis). The IgG1 heavy chain constant region Fc gamma 1, which plays an important role in infection resistance and has the dual functions of activating complement and mediating phagocytosis, is preferred in the embodiments of the present invention.

In a preferred embodiment, the invention provides an Ad5-BPI-Fc γ 1 recombinant adenovirus comprising a nucleotide sequence encoding a fusion protein of BPI and Fc, the coding sequence having in order from 5' to 3: human BPI signal peptide coding sequence, BPI_1-199The coding sequence, the human immunoglobulin hinge region and the coding sequence for Fc γ 1 are preferably linked at the 5 'and 3' ends, respectively, with a CMV promoter and SV40poly a expression control elements. In one embodiment, the nucleotide sequence of the BPI-Fc γ 1 chimeric gene encoding the BPI-Fc γ 1 fusion protein is as set forth in SEQ ID NO: 1, preferably wherein BPI_1-199The coding sequence for amino acid residues 4-24 is modified to be SEQ ID NO: 2.

the Ad5-BPI-Fc gamma 1 recombinant adenovirus infected animal cell mediated expression BPI-Fc gamma 1 fusion protein (called BPI-Fc gamma 1 protein in the following examples and figures) can directly kill in vitro, quickly and efficiently kill drug-resistant gram-negative bacteria by activating complement and opsonophagocytosis, and kill multiple drug-resistant (carrying drug-resistant genes) gram-negative bacteria in fresh whole blood of mice and human (simulating blood serum complement and phagocyte environment in vivo); the Ad5-BPI-Fc gamma 1 recombinant adenovirus has succeeded in the animal treatment of in vivo multiple drug-resistant (carrying drug-resistant gene) gram-negative bacteria infection model.

The drug-resistant gram-negative bacteria exemplified in the examples of the present invention are as follows:

1.E.coli pBR322/BL21(DE3)

drug resistance gene (antibiotic resistance gene): ampR and tetR

Drug resistance: ampicillin and tetracyline

2.E.coli pET28a-EGFP/BL21(DE3)

Drug resistance gene: kanR

Drug resistance: kanamycins

3. Acinetobacter baumannii (Acinetobacter baumannii) ATCC BAA-1605 (multiple drug resistance)

Drug resistance: ceftazidime, gentamicin, tiarcillin, piperacillin, aztreonam, cefepime, ciprofloxacin, imipenem, and meropemem

4. Klebsiella pneumoniae (Klebsiella pneumoniae) ATCC 700603 (with pod membrane) (multiple drug resistance, ESBLs)

Drug resistance gene: klebsiella pneumoniae plasma-encoded extended-specific beta-lactamase (bla SHV-18) gene

Drug resistance: ampicilin, aztreonam, cefoxitin, cefpodoxime, ceftazidime, chloremphenicol, piperacillin, tetracycline

5. Escherichia coli (NDM-1) ATCC BAA-2469

Drug resistance gene: new Delhi metallo-beta-lactamase (NDM-1) (blaNDM-1 super drug resistance gene)

Drug resistance: carbepenem-resistant (imipenem and ertapenem)

The WHO published the first global antibiotic resistance "key pathogen" list, i.e., the list of 12 bacterial races (Families) that pose the greatest threat to human health, in 2017, at 2/27, in an effort to solve the increasingly serious problem of global antimicrobial drug resistance. WHO classifies the list into three categories of paramount importance, very important and intermediate importance based on the degree of urgent need for new antibiotics. The most important group includes some multi-drug resistant bacteria that pose particular threats in hospitals, nursing homes, and patients requiring care with ventilators and blood conduits, including acinetobacter, pseudomonas, and various enterobacteriaceae, including klebsiella, escherichia coli, serratia, and proteus species. These bacteria can cause serious and often fatal infections such as bloodstream infections and pneumonia, and have developed resistance to a large number of antibiotics, including carbapenems and the third generation cephalosporins, the best available antibiotics currently used to treat multidrug resistant bacteria. The second and third levels of the list contain other bacteria that are increasingly resistant and cause more common illnesses, such as gonorrhea and food poisoning by salmonella. The preferred drug-resistant gram-negative bacteria and the drug-resistant mechanism thereof in the embodiment of the invention conform to the above-mentioned extremely important classes and have broad representativeness of multiple drug resistance (including drug-resistant genes) of each class; wherein the multiple resistance involves: resistant to ampicilin, tetracycline (tetracyclines), kanamycin (aminoglycosides), aztreonam, cefoxitin, cefpodoxime, ceftazidime, chloremphenicol, piperacillin, gentamicin (aminoglycosides), tiarcillin, cefepime, cipenem, ertapenem and carbepenem; the drug resistance gene relates to: ampR, tetR, kanR, extended-specific beta-lactamase bla SHV-18, New Delhi metal-beta-lactamase NDM-1.

In conclusion, the recombinant adenovirus of the invention has wide application as a drug-resistant gram-negative bacteria infection treatment drug. According to the characteristic that the adenovirus vector-mediated target gene is continuously expressed in vivo for 2-3 weeks, the recombinant adenovirus provided by the invention is used as a drug-resistant gram-negative bacteria infection medium-short-term antibiotic substitution treatment drug and a low-immune function patient medium-short-term gram-negative bacteria infection prevention drug, and has huge advantages and application prospects.

Drawings

FIG. 1 shows the liquid chromatography purification map of Ad5-BPI-Fc gamma 1 recombinant adenovirus. Wherein: A. SOURCE 30Q anion exchange chromatography; B. capto^TMCore 700 complex mode dielectric chromatography profile; C. capto^TMCore 700 complex mode media chromatography CIP clean-in-place profile.

FIG. 2 shows the liquid chromatography purification and identification results of BPI-Fc gamma 1 protein. Wherein: A. SP

Fast Flow cation exchange chromatography; B. protein a affinity chromatography profile; c. And purifying the detection result of the BPI-Fc gamma 1 protein Western Blot.

FIG. 3 BPI-Fc γ 1 protein binds LPS in vitro. Wherein: A. detecting the result by a gel semi-quantitative method; B. and (5) quantitatively detecting the result by an end point color development method.

FIG. 4 BPI-Fc gamma 1 protein in vitro killing and enhanced killing of drug resistant gram-negative bacteria by activating complement. Wherein: A. sterilizing in vitro and enhancing the sterilizing effect of the mouse serum complement; B. in vitro sterilization and human serum complement enhanced sterilization.

FIG. 5 shows the tracing experiment of BPI-Fc gamma 1 protein in vitro opsonization of BALB/c mouse peritoneal phagocytic cells E.coli pET28a-EGFP/BL21(DE 3). Wherein: A. phagocytosis (fluorescent) tracer assay. B. And counting results of phagocytosis phenomena in each group of experiments.

FIG. 6 shows the tracing experiment of BPI-Fc gamma 1 protein in vitro opsonization of human monocyte cell line U937 phagocytosis E. coli pET28a-EGFP/BL21(DE 3).

FIG. 7 shows the tracing experiment of BPI-Fc gamma 1 protein in vitro opsonization of human peripheral blood leukocyte by E.coli pET28a-EGFP/BL21(DE 3).

FIG. 8 BPI-Fc γ 1 protein opsonophagocytic killing in vitro E.coli pBR322/BL21(DE 3). Wherein: A. phagocytosis and sterilization results of abdominal phagocytes of BALB/c mice; B. the U937 cells phagocytose the bactericidal result.

FIG. 9 BPI-Fc γ 1 protein kills drug-resistant gene E.coli pBR322/BL21 in whole blood (DE 3).

FIG. 10 BPI-Fc γ 1 protein kills multiple drug-resistant A.baumannii in whole blood. Wherein: A. a mouse whole blood experiment; B. human whole blood experiments.

FIG. 11 BPI-Fc gamma 1 protein in whole blood kill multiple drug resistant (including drug resistant gene) Klebsiella pneumoniae. Wherein: A. a mouse whole blood experiment; B. human whole blood experiments.

FIG. 12 BPI-Fc γ 1 protein kills the super drug resistance gene Escherichia coli (NDM-1) in whole blood.

FIG. 13 Western Blot detection result of the expression of BPI-Fc gamma 1 protein in the serum of Ad5-BPI-Fc gamma 1 infected mice.

The technical solution of the present invention is further described below with reference to the following examples and drawings, but is not limited to the examples. More particularly, the first embodiment relates to the construction of Ad5-BPI-Fc gamma 1 recombinant adenovirus. The second embodiment relates to Ad5-BPI-Fc gamma 1 recombinant adenovirus mediated mammalian cell expression of BPI-Fc gamma 1 protein. Example three relates to direct killing of BPI-Fc γ 1 protein in vitro and enhanced killing of drug-resistant gram-negative bacteria by activating complement and opsonophagocytosis. Example four relates to the killing of drug-resistant gram-negative bacteria by the BPI-Fc gamma 1 protein in whole mouse and human blood. Example five relates to Ad5-BPI-Fc gamma 1 recombinant viruses on resistant gram negative bacteria infection model animal protection.

Example construction of an Ad5-BPI-Fc γ 1 recombinant adenovirus comprising a BPI-Fc chimeric Gene

Construction of pDC316-BPI-Fc gamma 1 adenovirus shuttle expression vector

According to the conventional molecular cloning experimental technology, the plasmid pSCm-BPIm23-Fc gamma 1 (constructed by the inventor) is subjected to double enzyme digestion by EcoR I/Sal I (wherein the EcoR I part is subjected to enzyme digestion), and a recovered 1.39kb EcoR I/Sal I enzyme digestion fragment (a BPI-Fc gamma 1 chimeric gene, the sequence of which is shown as SEQ ID NO: 1, and the fragment has a sequence from 5 'to 3' sequentially encoding a human BPI signal peptide and BPI_1-199Nucleotide sequences of human immunoglobulin hinge region and Fc γ 1; wherein BPI_1-199The natural coding sequence of amino acid residues 4-24 is modified by gene synthesis to the amino acid sequence of SEQ ID NO: 2) (ii) a Inserting the recovered enzyme digestion fragment construction into an EcoR I/Sal I enzyme digestion site of a pDC316 shuttle plasmid (Microbix) and transforming the enzyme digestion site to E.coli DH5 alpha; and identifying and correctly constructing to obtain a shuttle expression vector pDC316-BPI-Fc gamma 1.

The shuttle expression vector pDC316-BPI-Fc gamma 1 is already preserved in China general microbiological culture Collection center (CGMCC, No. 3 of Xilu 1 of North Cheng of the sunward area in Beijing of China) in 2018 at 11 and 8 months, and the preservation number is CGMCC No.: 16719, and is classified and named Escherichia coli.

Packaging and preparation of Ad5-BPI-Fc gamma 1 recombinant adenovirus

Briefly described as follows: reference AdMax^TMSystem (kit D) Manual (Microbix) by convention

2000 transfection method HEK 293 cells (ATCC CRL-1573) were co-transfected by pDC316-BPI-Fc gamma 1 shuttle expression vector and adenovirus backbone vector pBHGlox (delta) E1,3Cre, and recombinant generation of Ad5-BPI-Fc gamma 1 recombinant adenovirus was achieved by Cre/loxP system. Under GMP compliant conditions: 1) collecting virus plaques, performing virus amplification and amplification production (a)

3205L tank bioreactor, Fibra-

The sheet-like carriers were inoculated with 10% NBS-containing DMEM growth medium before inoculation and with 10% NBS-containing DMEM after inoculationDMEM maintenance medium with 2% NBS perfused to culture HEK 293 cells); 2) viral supernatants were collected by centrifugation at 8000rpm, prefiltered by tangential flow using a 0.65 μm PVDF membrane cartridge (Millipore),

2, concentrating by using a box type membrane (300K) ultrafiltration membrane, and treating by using Benzonase nuclease; 3) subjecting to SOURCE 30Q anion exchange chromatography (as shown in FIG. 1A) and Capto^TMSeparating and purifying Core 700 composite mode medium chromatography (GE Life Sciences) (as shown in figure 1B; and then cleaning CIP in situ as shown in figure 1C), and collecting the peak eluted by Ad5-BPI-Fc gamma 1; 4) concentration and replacement of Virus stocks (10mM Tris 150mM NaCl 1mM MgCl) with Vivaflow 200100K Ultrafiltration Membrane pack (Sartorius)₂10% glycerol pH7.4), obtaining high-quality Ad5-BPI-Fc gamma 1 recombinant adenovirus, and storing at-70 ℃ for later use. Through detection: the virus titer is more than or equal to 5 multiplied by 10⁹IU/mL, specific titer (IU/VP) is more than or equal to 3.3% (TCID 50 and OD260 detection respectively), and OD260/280 ratio is 1.2-1.4; after PCR amplification, the target BPI-Fc gamma 1 chimeric gene is subjected to DNA sequencing to show that the sequence of the target BPI-Fc gamma 1 chimeric gene is similar to the sequence shown in SEQ ID NO: 1, and then, mixing.

Example Ad5-BPI-Fc gamma 1 recombinant adenovirus mediated mammalian cell expression of BPI-Fc gamma 1 protein and purification thereof

CELLSPIN&The protein flash (INTEGRAbiosciences AG) was inoculated with CHO-DG44 cells (Gibco) and Ad5-BPI-Fc γ 1 recombinant adenovirus (MOI ═ 40) (in serum-free Medium CD DG44Medium, 37 ℃, 8% CO₂Standing for 2h), adding appropriate amount of SP

Fast Flow Co-culture for 5 days (60rpm), SP was collected

Fast Flow and packing the Flow into a column for chromatographic separation and purification (as shown in FIG. 2A); then separating and purifying by Protein A affinity chromatography (as shown in figure 2B), and collecting BPI-Fc gamma 1 Protein elution peak; concentration by Amicon Ultra-15(NMWL 30KD) ultrafiltration centrifugation and displacement of protein stock solution (0.15M NaCl 20mM citric acid 0.1% v/v Poloxamer 1880.002% v/v Polysorbate 80pH 5.0) is stored at-30 ℃ until use. Identified by Western BlotAs shown in FIG. 2C, the expected 48kDa band appears in the DTT reduced lane, and the main band is 96kDa in the non-DTT reduced lane.

Example biological function of the three BPI-Fc γ 1 proteins: in vitro binding of LPS, direct killing and enhanced killing of drug-resistant gram-negative bacteria by activating complement and opsonophagocytosis.

Coli pBR322/BL21(DE3) (selected by ampicillin) and E.coli pET28a-EGFP/BL21(DE3) (selected by kanamycin and induced by 1mM IPTG at 30 ℃ for 16h to express EGFP) were used for in vitro killing of drug-resistant gram-negative bacteria.

BPI-Fc gamma 1 protein binding to LPS in vitro

1.1 Add 200. mu.L of goat anti-human IgG-Fc (Novex) diluted with 0.5. mu.g/mL PBS per well in 96-well ELISA plates without endotoxin, coat overnight at 4 ℃ and wash the plates with PBST (PBS containing 0.1% Tween-20) (5 min/time, 3 times total); then 200. mu.L of PBST solution containing 5% BSA was added to each well, blocked at 37 ℃ for 1h, and the plate was washed with PBST (5 min/time, 3 times in total); add 200. mu.L of an incubation mixture of BPI-Fc γ 1 protein (0. mu.g/mL and 8. mu.g/mL) and LPS (endotoxin standard, 8EU/mL) to each well (vortex mix, 37 ℃,30min) and incubate for 1h at 37 ℃. The gel semiquantitative assay was performed according to the instruction manual of Limulus reagent (Tachypleus tridentatus Biotech Co., Ltd.). The results are shown in FIG. 3A: the BPI-Fc gamma 1 protein can effectively bind LPS.

1.2 Add 200. mu.L of goat anti-human IgG-Fc (Novex) diluted with 0.5. mu.g/mL PBS per well in 96-well ELISA plate without endotoxin, coat overnight at 4 ℃ and wash plate with PBST (5 min/time, total 3 times); then 200. mu.L of PBST solution containing 5% BSA was added to each well, blocked at 37 ℃ for 1h, and the plate was washed with PBST (5 min/time, 3 times in total); mu.L of incubation mixture (vortexed, 37 ℃ C., 30min) containing varying concentrations of BPI-Fc γ 1 protein (0.025. mu.g/mL and 0.1. mu.g/mL; both negative and positive controls were replaced with equal amounts of endotoxin-free PBS) and LPS (endotoxin standard, 0.05 EU/mL; negative controls were replaced with water for bacterial endotoxin testing) was added to each well and incubated for 1h at 37 ℃. 100. mu.L of each well was incubated and samples were expressed as ToxinSensors^TMEndotoxin test kit (Nanjing Kingsrei Biotech Co., Ltd., L00350) instruction bookColor reaction is carried out, and the OD545nm value is determined and the endotoxin content in the sample is calculated. The results are shown in FIG. 3B: the BPI-Fc gamma 1 protein can effectively bind to LPS, and the binding capacity of the protein presents a dose-dependent relationship.

BPI-Fc gamma 1 protein in vitro sterilization and drug-resistant gram-negative bacteria killing by activating complement

2.1 BPI-Fc gamma 1 protein in vitro sterilization and mouse serum complement enhanced sterilization

50 μ L of E.coli pBR322/BL21(DE3) bacterial liquid (2.5 × 10)⁵CFU/mL, diluted in 10% Hanks 40mM Tris-HCl 0.1% casamino acid pH 7.5; the negative control was replaced with the same amount of the buffer) and 50. mu.L of BPI-Fc γ 1 protein (0.8. mu.g/mL, 0.4. mu.g/mL, 0.2. mu.g/mL) were diluted in a protein stock; negative and positive controls are replaced by equal amount of protein preservation solution) and mixed evenly, and water bath is carried out at 37 ℃ for 3 h; then 100. mu.L of 4% BALB/c mouse serum (PBS dilution; control group is replaced by PBS with equal amount) is added, and water bath is carried out for 1h at 37 ℃; and (5) diluting the bacterial liquid and counting by a retrogradation injection method. The results are shown in FIG. 4A: the BPI-Fc gamma 1 protein can effectively kill gram-negative bacteria in vitro, and can enhance the bactericidal effect by cross-acting with the mouse serum complement and activating the mouse complement, and the bactericidal efficiency and the dosage are positively correlated.

2.2 BPI-Fc gamma 1 protein in vitro sterilization and human serum complement enhanced sterilization

The procedure was as described above (2.1), and an experiment was carried out using 4% human serum (while changing the BPI-Fc. gamma.1 protein concentration to 1. mu.g/mL, 0.5. mu.g/mL, 0.1. mu.g/mL). The results are shown in FIG. 4B: the BPI-Fc gamma 1 protein can effectively kill gram-negative bacteria in vitro, can enhance the bactericidal effect by activating human serum complement, and has the bactericidal efficiency positively correlated with the dosage.

Killing drug-resistant gram-negative bacteria by opsonophagocytosis in vitro with BPI-Fc gamma 1 protein

3.1 BPI-Fc gamma 1 protein opsonophagocytosis drug-resistant gram-negative bacteria tracing experiment

3.1.1 isolation of BALB/c mouse peritoneal phagocytes: killing the mouse after removing the vertebra, and soaking the mouse in 75% ethanol for 5-10 min; removing the skin and hair of the abdominal cavity of the mouse and keeping the peritoneal intact; injecting a proper amount of DMEM medium into the abdominal cavity of the mouse, and gently massaging for 5 min; the mouse peritoneal cell suspension was extracted, 30Centrifuging for 5min at 0g to collect cells; complete medium (DMEM medium with 10% NBS) was resuspended and inoculated into 24-well plates (containing cell-slides) at 37 ℃ with 8% CO₂Culturing for 3-7 days. Staining with DiI (Beyotime) for 20min, and washing with PBS 3 times; 50 μ L of E.coli pET28a-EGFP/BL21(DE3) bacterial solution (PBS was washed and prepared to 2X 10⁹CFU/mL suspension; negative control was replaced with equal amount of PBS) and 50 μ L BPI-Fc γ 1 protein (20 μ g/mL, diluted in protein stock; negative and positive controls are replaced by equal amount of protein preservation solution), mixing, incubating at 37 deg.C for 10min, covering on cells, and incubating at 37 deg.C for 60 min; PBS washing 3 times, fixing with fixing solution (acetic acid: methanol 1:3 mixture) for 5min, PBS washing 3 times, placing the slide on the slide, sealing, and observing under fluorescence microscope. The results are shown in FIG. 5A: DiI identifies mouse abdominal cavity phagocytes (DiI, red fluorescence), induced expression EGFP identifies e.coli pET28a-EGFP/BL21(DE3) (EGFP, green fluorescence), and two identification image superposition treatments (DiI + EGFP) are performed, wherein the arrow head indicates phagocytosis phenomenon (cell membrane or intracellular EGFP green fluorescence), it can be seen that BPI-Fc γ 1 protein can cross with mouse species Fc receptor and obviously promote mouse phagocytes to phagocytize e e.coli pET28a-EGFP/BL21(DE 3); FIG. 5B shows that the addition of BPI-Fc γ 1 protein promoted an increase in phagocytosis from 25% to 92.5%.

3.1.2 Collection of human monocytic cell line U937 cells (Shanghai Life sciences research institute of Chinese academy of sciences, cell resource center, 4X 10)⁶cells) were stained with DiI for 30min and washed 2 times with PBS for future use; 110. mu.L of E.coli pET28a-EGFP/BL21(DE3) bacterial solution (PBS was washed and prepared to 2X 10⁹CFU/mL suspension; negative control replaced with equal amount of PBS) and 110 μ L BPI-Fc γ 1 protein (40 μ g/mL, diluted in protein stock; negative and positive controls were replaced with equal amounts of protein stock solution) and mixed well, incubated at 37 ℃ for 20 min; mixing 200 μ L of the mixture with U937 cells, incubating at 37 deg.C and 150rpm for 2.5 h; centrifuging for 1min at 200g, discarding the supernatant, and washing with PBS for 2 times; fixing with 4% tissue cell fixing solution (solarBio) for 10 min; PBS and ultrapure water are washed for 1 time in sequence, a proper amount of mounting solution is taken to suspend cells again, then the cells are dripped on a glass slide, and mounting and observation are carried out under a fluorescent microscope. The results are shown in FIG. 6: the identification is as above (3.1.1), and BPI-Fc can be seenThe gamma 1 protein can remarkably promote U937 cells to phagocytose E.coli pET28a-EGFP/BL21(DE 3).

3.1.3 isolation and Collection of human leukocytes (4X 10) Using the human peripheral blood leukocyte fraction kit (solarBio)⁶cells). The method is as above (3.1.2), wherein it becomes: 150 μ L of the mixture was mixed with human leukocyte and incubated at 37 ℃ for 1h with shaking at 200 rpm. The results are shown in FIG. 7: as identified above (3.1.1), since human leukocytes are non-single constituent cells, DiI staining effects and fluorescence quenching times are different among different types of phagocytes, DiI does not uniformly identify all phagocytes, and therefore visible light images are added for comparison, as shown in fig. 7: BPI-Fc gamma 1 protein can significantly promote human leukocyte phagocytosis E.coli pET28a-EGFP/BL21(DE 3).

3.2 killing of drug-resistant gram-negative bacteria by opsonophagocytosis of BPI-Fc gamma 1 protein in vitro

1) BALB/c mouse peritoneal phagocytes were isolated (as above 3.1.1) and plated in 96-well plates at 37 ℃ with 8% CO₂Culturing for 1-3 days (confluency)>80%); 30 μ L of E.coli pBR322/BL21(DE3) bacterial solution (PBS washed and prepared 2X 10⁷CFU/mL suspension; negative control with equal amount of PBS replacement) and 30. mu.L of BPI-Fc gamma 1 protein (4. mu.g/mL, 2. mu.g/mL, diluted in protein stock; negative and positive controls are respectively replaced by equivalent protein preservation solution), evenly mixed, bathed in water at 37 ℃ for 10min, evenly covered on cells, and incubated at 37 ℃ for 60 min; after each incubation sample was mixed well, 20. mu.L of each incubation sample was diluted by a double ratio (10)^-1～10^-5) Taking 100 mu L of each diluted sample, and counting colonies by a pouring method; the above procedure was carried out using CHO-DG44 cells as a control.

2) Collection of human monocyte cell line U937 (2X 10)⁶cells), PBS washed 2 times for use; 60 μ L of E.coli pBR322/BL21(DE3) was washed with PBS and prepared into 2X 10⁷CFU/mL suspension; negative control with equal amount of PBS replacement) and 60. mu.L of BPI-Fc gamma 1 protein (2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, diluted in protein stock; negative and positive control are respectively replaced by equal amount of protein preservation solution), mixing, and water bathing at 37 deg.C for 20 min; mixing 100 μ L of the mixture with U937 cells, and incubating at 37 ℃ with shaking at 200rpm for 1h (in a 96-well plate); 300g, 5 g of each incubated sampleThe supernatant was collected by min centrifugation and 20. mu.L each was diluted by a factor of 10^-1～10^-5) Taking 100 mu L of each diluted sample respectively, and counting colonies by a pouring method; the above procedure was carried out using CHO-DG44 cells as a control.

The results are shown in FIG. 8: the BPI-Fc gamma 1 protein can phagocytose and kill drug-resistant gram-negative bacteria in vitro by opsonizing mouse abdominal cavity phagocytic cells (figure 8A) and U937 cells (figure 8B), and the phagocytic sterilization rate is in positive correlation with the concentration of the BPI-Fc gamma 1 protein.

EXAMPLE four BPI-Fc γ 1 proteins kill drug-resistant gram-negative bacteria in Whole blood

Research on killing of drug-resistant gram-negative bacteria by fresh whole blood (containing serum complement and phagocytes) was carried out by colii pBR322/BL21(DE3), Acinetobacter baumannii ATCC BAA-1605, Klebsiella pneumoniae ATCC 700603 (with capsule) and Escherichia coli (NDM-1) ATCC BAA-2469 (selectively cultured with ampicillin, imipenem, cefoxitin and imipenem, respectively).

BPI-Fc gamma 1 protein kills drug-resistant gene E.coli pBR322/BL21 in whole blood (DE3)

In view of the strong resistance (e.g., serotype response and phagocytic clearance) of fresh human whole blood to e.coli pBR322/BL21(DE3), the study was conducted to kill e.coli pBR322/BL21(DE3) in mouse whole blood.

20 μ L of E.coli pBR322/BL21(DE3) bacterial liquid (5 × 10)⁵CFU/mL, PBS dilution; negative control with equal amount of PBS replacement) and 20. mu.L of BPI-Fc gamma 1 protein (5. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL) at different concentrations, diluted in protein stock; negative and positive control are respectively replaced by equivalent protein preservation solution), mixing, and incubating at 37 deg.C for 10 min; adding 80 μ L of fresh 0.4% sodium citrate anticoagulated BABL/c mouse whole blood, mixing well and incubating at 37 ℃ for 3 h; after appropriate dilution, 100. mu.L of each was counted by decantation. The results are shown in FIG. 9: the BPI-Fc gamma 1 protein has obvious killing effect on a drug-resistant gene E.coli pBR322/BL21(DE3) in whole blood, and the sterilization efficiency of the BPI-Fc gamma 1 protein is positively correlated with the protein dose; wherein when the protein concentration is 0.5 and 5 mu g/mL, the corresponding sterilization rates are 71.5 percent and 99.5 percent respectively. The results of FIG. 4 and FIG. 8 show that BPI-Fc gamma 1 protein can be activated in whole bloodComplement and opsonophagocytosis kill gram-negative bacteria efficiently.

Killing multiple drug-resistant Acinetobacter baumannii (ATCC BAA-1605) with BPI-Fc γ 1 protein in whole blood

2.1 killing Acinetobacter baumannii in Whole blood of mice

Collecting 20 μ L Acinetobacter baumannii (ATCC BAA-1605) bacterial solution (1 × 10)⁴CFU/mL) and 20 μ L of BPI-Fc γ 1 protein (50 μ g/mL, 5 μ g/mL, 0.5 μ g/mL, diluted in protein stock solution; negative and positive control are respectively replaced by equivalent protein preservation solution), mixing, and incubating at 37 deg.C for 10 min; adding 80 μ L of fresh 0.4% sodium citrate anticoagulated BABL/c mouse whole blood, mixing well and incubating at 37 ℃ for 1 h; after appropriate dilution, 100. mu.L of each was counted by decantation. The results are shown in FIG. 10A: the BPI-Fc gamma 1 protein has obvious killing effect on multiple drug-resistant acinetobacter baumannii in whole blood of a mouse, and the sterilization efficiency of the BPI-Fc gamma 1 protein is positively correlated with the protein dose; wherein when the protein concentration is 50 mug/mL, the corresponding sterilization rate is 25.4%.

2.2 killing Acinetobacter baumannii in human Whole blood

The procedure was as above (2.1) and experiments were carried out with human blood. The results are shown in FIG. 10B: the BPI-Fc gamma 1 protein has obvious killing effect on multiple drug-resistant acinetobacter baumannii in human whole blood, and the sterilization efficiency of the BPI-Fc gamma 1 protein is positively correlated with the protein dose; wherein when the protein concentration is 0.5, 5 and 50 mug/mL, the corresponding sterilization rate respectively reaches 6.9 percent, 13.2 percent and 43.4 percent.

Killing of multidrug (including resistance genes) resistant Klebsiella pneumoniae (ATCC 700603) with BPI-Fc gamma 1 protein in whole blood

3.1 killing Klebsiella pneumoniae in Whole blood of mice

Collecting 50 μ L Klebsiella pneumoniae (ATCC 700603) (1 × 10)⁴CFU/mL, PBS dilution; negative control with equal amount of PBS replacement) and 50. mu.L of BPI-Fc gamma 1 protein (1000. mu.g/mL, 500. mu.g/mL, 250. mu.g/mL) at different concentrations, diluted in protein stock; negative and positive controls were replaced with equivalent amounts of protein preservation solutions, respectively) were mixed well and incubated at 37 ℃ for 3 h; 100 μ L of fresh 0.4% sodium citrate anticoagulated BABL/c mouse whole blood was added, mixed well and incubated at 37 ℃ for 1 h; after appropriate dilution, 100. mu.L of each was counted by decantation. The results are as followsFIG. 11A shows: when the BPI-Fc gamma 1 protein reaches higher effective concentration, the multi-drug resistant (including drug resistant gene) Klebsiella pneumoniae with capsule (resisting phagocytosis and preventing BPI N-terminal functional fragment from being combined with LPS) in whole blood of a mouse has obvious killing effect, and the sterilization efficiency and the protein dosage are positively correlated; wherein when the concentration of the protein is 500 and 1000 mug/mL, the corresponding sterilization rates respectively reach 82.5 percent and 98.3 percent.

3.2 killing Klebsiella pneumoniae in human Whole blood

The procedure was as above (3.1) and experiments were carried out with human blood. The results are shown in FIG. 11B: with the killing effect on the whole blood of a mouse, when the BPI-Fc gamma 1 protein reaches the effective concentration, the BPI-Fc gamma 1 protein has an obvious killing effect on multiple drug-resistant (including drug-resistant genes) Klebsiella pneumoniae in the whole blood of a human, and the sterilization efficiency and the protein dose are positively correlated; wherein when the protein concentration is 500 and 1000 mug/mL, the corresponding sterilization rate respectively reaches 76.7 percent and 94.0 percent.

Killing of super drug-resistant Gene Escherichia coli (NDM-1) with BPI-Fc γ 1 protein in Whole blood

In view of the strong resistance (e.g., serotype response and phagocytic clearance) of fresh human whole blood to Escherichia coli (NDM-1) (healthy volunteers), the present study was conducted to kill Escherichia coli (NDM-1) in whole mouse blood.

mu.L of Escherichia coli (NDM-1) (ATCC BAA-2469) (1X 10)⁴CFU/mL) and 20 μ L of BPI-Fc γ 1 protein (50 μ g/mL, 5 μ g/mL, 0.5 μ g/mL, diluted in protein stock solution; negative and positive controls were replaced with equivalent amounts of protein preservation solutions, respectively) were mixed well and incubated at 37 ℃ for 1 h; adding 80 μ L of fresh 0.4% sodium citrate anticoagulated BABL/c mouse whole blood, mixing well and incubating at 37 ℃ for 1 h; after appropriate dilution, 100. mu.L of each was counted by decantation. The results are shown in FIG. 12: the BPI-Fc gamma 1 protein has obvious killing effect on super drug-resistant gene Escherichia coli (NDM-1) in whole blood, and the sterilization efficiency of the BPI-Fc gamma 1 protein is positively correlated with the protein dose; wherein when the protein concentration is 5 and 50 mu g/mL, the corresponding bactericidal rate respectively reaches 21.8 percent and 70.7 percent.

Example five Ad5-BPI-Fc gamma 1 recombinant viruses mediate the expression of BPI-Fc gamma 1 protein in mice and the protection effect on drug-resistant gram-negative bacteria infection model animals

Ad5-BPI-Fc gamma 1 recombinant virus mediated BPI-Fc gamma 1 protein expression in mouse serum

5-6 week-old BALB/c mice were injected with Ad5-BPI-Fc gamma 1 recombinant virus (5X 10)⁸IU/100 uL/each) (Experimental Ad5-Null control group), and blood was collected from the eye on day 3 and day 5 after virus injection to obtain serum. The serum is treated by saturated ammonium sulfate precipitation to obtain crude immunoglobulin precipitate, which is briefly as follows: 1) adding equivalent physiological saline into 0.5mL of serum, mixing, dropwise adding saturated ammonium sulfate 1mL under stirring, standing at 4 deg.C for 1h, centrifuging at 3000rpm for 20min, and removing supernatant; 2) dissolving the precipitate with normal saline to 1mL, dropwise adding saturated ammonium sulfate 0.5mL, standing at 4 deg.C for 3h, centrifuging at 3000rpm for 20min, and removing the supernatant; 3) repeating 2) for 1 time to obtain crude immunoglobulin precipitate; 4) dissolving the serum immunoglobulin in 1mL of PBS, transferring the solution into a dialysis bag, fully dialyzing the solution with the PBS, and embedding the dialysis bag into polyethylene glycol to absorb water and concentrate the solution to 50ul of serum immunoglobulin concentrated solution for later use. And (3) conventional ELISA detection: human IgG ELISA kit (1750) (purchased from Alpha Diagnostic); the results show that: BPI-Fc γ 1 protein expression levels in mouse sera were 21.4 ± 0.2ng/mL (n ═ 3) and 30.5 ± 7.4ng/mL (n ═ 3), respectively, on days 3 and 5 after Ad5-BPI-Fc γ 1 injection (note: no detection in Ad5-Null control group). Conventional Western Blot detection: detecting goat anti-human IgG Fc polyclonal antibody (purchased from KPL company, UK) marked by HRP, and exposing and developing by X-ray film according to the operation instruction of a Western Blot luminescence kit (purchased from Pierce); the results are shown in FIG. 13: the expected Ad5-BPI-Fc γ 1 protein band (non-reducing) was expressed in serum from mice infected with Ad5-BPI-Fc γ 1 at 96kD, whereas the Ad5-Null control group did not (note: the band appearing at 140kD is considered to be the band where goat anti-human IgG Fc polyclonal antibodies cross mouse IgG).

Protection of Ad5-BPI-Fc gamma 1 recombinant viruses against drug-resistant gram-negative bacteria infecting mice

Coli pBR322/BL21(DE3) (ampR and tetR genes) and klebsiella pneumoniae (ATCC 700603) (selectively cultured with ampicillin and cefoxitin, respectively) were selected from the drug-resistant gram-negative bacteria referred to in the preceding examples for study.

2.1 minimum lethality assay of drug-resistant gram-negative bacteria infected BALB/c mice

Using high-activity dry yeast containing 5%

Diluting drug-resistant gram-negative bacteria to be detected into bacteria liquids with different concentrations by PBS, injecting the bacteria liquids (0.5 mL/each) into abdominal cavities of BALB/c mice of 6-7 weeks old which are randomly grouped (10 mice in each group), and observing the death condition of the mice; the minimum amount of bacteria causing 90-100% of infected deaths in 72 hours was determined as the Minimum Lethal Dose (MLD). And (3) experimental determination: coli pBR322/BL21(DE3) has an intraperitoneal MLD of 2.5X 10⁴CFU/0.5 mL; the MLD of Klebsiella pneumoniae (ATCC 700603) by intraperitoneal injection is 1X 10⁶CFU/0.5mL or 2.5X 10⁶CFU/0.5mL。

2.2 protection of Ad5-BPI-Fc gamma 1 recombinant viruses against infection of mice with the drug resistance gene E.coli pBR322/BL21(DE3)

Randomly grouped (10 mice per group) 6-7 week old BALB/c mice were injected intraperitoneally with 5X 10 cells⁸IU/100. mu.L Ad5-BPI-Fc gamma 1 recombinant virus (PBS control group and Ad5-Null control group are used for experiments), MLD lethal dose drug-resistant gram-negative bacteria are injected into the abdominal cavity of each mouse on the 3 rd day for infection and challenge, and the death condition of the mouse is observed within 72 hours. The results are shown in table 1: ad5-BPI-Fc gamma 1 recombinant virus transfected mice have significant protective effect against lethal dose resistance gene E.coli pBR322/BL21(DE3) (ampR and tetR genes) infection attack.

TABLE 1 protection against lethal dose resistance gene E.coli pBR322/BL21(DE3) infection challenge

(compared to Ad5-Null and PBS groups:^#P<0.05)

2.3 protection action of Ad5-BPI-Fc gamma 1 recombinant virus on mice infected by multidrug-resistant Klebsiella pneumoniae

The procedure was as above (2.2), and the results are shown in Table 2: the Ad5-BPI-Fc gamma 1 recombinant virus transfected mice have obvious protection effect on lethal dose multiple drug resistance (including drug resistance genes) Klebsiella pneumoniae (ATCC 700603) infection attack.

TABLE 2 protective Effect against lethal multiple drug-resistant challenge of Klebsiella pneumoniae infection

Note: MLD of 1 × 10⁶CFU/0.5mL,. times.MLD 2.5 × 10⁶CFU/0.5mL。

(compared to the Ad5-BPI-Fc γ 1 group:^#P<0.05，^##P<0.01)。

sequence listing

<110> Xiamen Union Anjin bioengineering Co., Ltd

Anjun (Beijing) Gene science and technology Limited liability company

<120> recombinant adenovirus containing BPI-Fc chimeric gene and use thereof

<130> IDC180251

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1389

<212> DNA

<213> Artificial sequence

<400> 1

aattcggtac catgagagag aacatggcca ggggcccttg caacgcgccg agatgggtgt 60

ccctgatggt gctcgtcgcc ataggcaccg ccgtgacagc ggccgtcaac cctggtgttg 120

tagttcgtat ctctcagaaa ggtctggact acgcttctca gcaaggtact gctgcactgc 180

agaaggagct gaagaggatc aagattcctg actactcaga cagctttaag atcaagcatc 240

ttgggaaggg gcattatagc ttctacagca tggacatccg tgaattccag cttcccagtt 300

cccagataag catggtgccc aatgtgggcc ttaagttctc catcagcaac gccaatatca 360

agatcagcgg gaaatggaag gcacaaaaga gattcttaaa aatgagcggc aattttgacc 420

tgagcataga aggcatgtcc atttcggctg atctgaagct gggcagtaac cccacgtcag 480

gcaagcccac catcaccgcc tccagctgca gcagccacat caacagtgtc cacgtgcaca 540

tctcaaagag caaagtcggg tggctgatcc aactcttcca caaaaaaatt gagtctgcgc 600

ttcgaaacaa gatgaacagc caggtctgcg agaaagtgac caattctgta tcctccaagc 660

tgcaacctta tttccagact ctgccagtaa tgaccaaaat agaagcttct acatgcccac 720

cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc ccaaaaccca 780

aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg gacgtgagcc 840

acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg cataatgcca 900

agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg 960

tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc aacaaagccc 1020

tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga gaaccacagg 1080

tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc 1140

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agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc ttcctctaca 1260

gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca tgctccgtga 1320

tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct ccgggtaaat 1380

aaggatccg 1389

<210> 2

<211> 63

<212> DNA

<213> Artificial sequence

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gca 63

Claims

1. Use of a recombinant adenovirus in the manufacture of a medicament for the treatment of a drug-resistant gram-negative bacterial infection, wherein said drug-resistant gram-negative bacteria is Acinetobacter baumannii, wherein said recombinant adenovirus comprises a chimeric gene encoding a BPI-Fc fusion protein, wherein said chimeric gene comprises a coding sequence for a functional fragment of human BPI and a human immunoglobulin heavy chain constant region Fc gene, wherein the 3' end of the coding sequence for the functional fragment of human BPI is linked to the Fc gene by a human immunoglobulin hinge region, wherein the sequence of the chimeric gene encoding the BPI-Fc fusion protein is as set forth in SEQ ID NO: 1, and wherein said adenovirus is Ad5 serotype.

2. The use of claim 1, wherein the chimeric gene encoding a BPI-Fc fusion protein has, in order from 5 'to 3': human BPI signal peptide coding sequence, BPI_1-199Fragment coding sequence, human immunoglobulin hinge region and Fc γ 1 coding sequence, to which CMV promoter and SV40poly a expression control elements are attached at the 5 'end and 3' end, respectively.

3. The use according to any of claims 1-2, wherein the BPI_1-199The coding sequence of amino acid residues 4-24 of the fragment is SEQ ID NO: 2.