CN118620072A - Klebsiella pneumoniae polysaccharide monoclonal antibody, hybridoma cell line thereof and application thereof - Google Patents

Abstract

The present invention discloses a Klebsiella pneumoniae polysaccharide monoclonal antibody, a hybridoma cell line thereof and an application thereof. The sequence information of the Klebsiella pneumoniae polysaccharide monoclonal antibody is as follows: SEQ NO: 1 to SEQ NO:64, the deposit numbers of the hybridoma cell lines are CCTCC:C2023296, CCTCC:C2023311, CCTCC:C2023312, CCTCC:C2023313, CCTCC:C2023314, CCTCC:C2023365, CCTCC:C2023366 and CCTCC:C2023367, respectively. The Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention can specifically bind to the capsular polysaccharide of Klebsiella, which is beneficial to the typing of Klebsiella pneumoniae in clinical practice. In vivo and in vitro tests on animals, it can be proved that it has excellent effects in preventing and treating K47 and K64 type Klebsiella pneumoniae infections, provides a way to solve the problems of Klebsiella pneumoniae infection and multidrug resistance, has significant significance for the prevention, diagnosis and treatment of Klebsiella pneumoniae infection, and has important value for the development of new generation of Klebsiella pneumoniae drugs.

Description

A Klebsiella pneumoniae polysaccharide monoclonal antibody, its hybridoma cell line and its application

Technical Field

The invention belongs to the field of medical technology, and particularly relates to a Klebsiella pneumoniae polysaccharide monoclonal antibody, a hybridoma cell line thereof and applications thereof.

Background Art

Klebsiella pneumoniae is a Gram-negative bacterium (G-) with a capsule. Klebsiella pneumoniae can be divided into at least 77 serotypes according to the different structures of capsular polysaccharides, which are also called K antigens or K types. Klebsiella pneumoniae mainly colonizes the digestive tract and respiratory tract. When the body's resistance is reduced, it can cause lung infection, urinary tract infection, and even bloodstream infection. It is also one of the most common bacteria causing nosocomial respiratory tract infections. Capsular polysaccharides are the main virulence factors of Klebsiella pneumoniae and are important protective antigens for the treatment or prevention of Klebsiella pneumoniae infections.

Infections caused by Klebsiella pneumoniae have become one of the most serious public health problems in the world. According to statistics, the mortality rate of Klebsiella pneumoniae infection is extremely high. The mortality rate of bloodstream infection caused by Klebsiella pneumoniae is 20% to 30%, while the mortality rate of Klebsiella pneumoniae bacteremia combined with pneumonia can reach more than 50%. The treatment of Klebsiella pneumoniae mainly relies on antibiotics, but in recent years, due to the widespread use of various antimicrobial drugs, Klebsiella pneumoniae that produces extended-spectrum β-lactamases and carbapenemases are resistant to almost all available β-lactam drugs (including carbapenems). In the past 10 years, the rate of carbapenem-resistant Klebsiella pneumoniae (CRKP) has increased dramatically worldwide.

Therefore, there is a need in the art to develop a Klebsiella pneumoniae polysaccharide monoclonal antibody with high specificity, good affinity and strong binding ability, so as to facilitate the prevention and control of drug-resistant Klebsiella pneumoniae and its clinical application.

Summary of the invention

To solve the above technical problems, the present invention provides a Klebsiella pneumoniae polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K47 type Klebsiella pneumoniae, and the deposit number of the K47 type Klebsiella pneumoniae is GDMCC No: 62131. The sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 2.

Specifically, the complementarity determining region of the light chain variable region contains three sequences, as shown in SEQ ID: 3, SEQ ID: 4, and SEQ ID: 5, and the complementarity determining region of the heavy chain variable region contains three sequences, as shown in SEQ ID: 6, SEQ ID: 7, and SEQ ID: 8.

In a second aspect, the present invention provides a hybridoma cell strain, the hybridoma cell strain is named K47-5B9-F8, the deposit number of the hybridoma cell strain is: CCTCC NO: C2023314, and the hybridoma cell strain is used to prepare the aforementioned Klebsiella pneumoniae polysaccharide monoclonal antibody.

In a third aspect, the present invention provides a Klebsiella pneumoniae polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K47 type Klebsiella pneumoniae, and the preservation number of the K47 type Klebsiella pneumoniae is GDMCC No: 62131. The sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 9, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 10.

Specifically, the complementary determining region of the light chain variable region contains three sequences, as shown in SEQ ID: 11, SEQ ID: 12, and SEQ ID: 13, and the complementary determining region of the heavy chain variable region contains three sequences, as shown in SEQ ID: 14, SEQ ID: 15, and SEQ ID: 16.

In a fourth aspect, the present invention provides a hybridoma cell strain, the hybridoma cell strain is named K47-15D7-B10, the deposit number of the hybridoma cell strain is: CCTCC NO: C2023312, and the hybridoma cell strain is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody described in the third aspect.

In a fifth aspect, the present invention provides a Klebsiella pneumoniae polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K47 type Klebsiella pneumoniae, and the deposit number of the K47 type Klebsiella pneumoniae is GDMCC No: 62131. The sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 17, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 18.

Specifically, the complementarity determining region of the light chain variable region contains three sequences, as shown in SEQ ID: 19, SEQ ID: 20, and SEQ ID: 21, and the complementarity determining region of the heavy chain variable region contains three sequences, as shown in SEQ ID: 22, SEQ ID: 23, and SEQ ID: 24.

In a sixth aspect, the present invention provides a hybridoma cell strain, which is named K47-18G6-B2, and the deposit number of the hybridoma cell strain is: CCTCC NO: C2023311. The hybridoma cell strain is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody described in the fifth aspect.

In a seventh aspect, the present invention provides a Klebsiella pneumoniae polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K47 type Klebsiella pneumoniae, and the deposit number of the K47 type Klebsiella pneumoniae is GDMCC No: 62131. The sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 25, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 26.

Specifically, the complementarity determining region of the light chain variable region contains three sequences, as shown in SEQ ID: 27, SEQ ID: 28 and SEQ ID: 29, and the complementarity determining region of the heavy chain variable region contains three sequences, as shown in SEQ ID: 30, SEQ ID: 31 and SEQ ID: 32.

In an eighth aspect, the present invention provides a hybridoma cell strain, which is named K47-6E2, and the deposit number of the hybridoma cell strain is: CCTCC NO: C2023296. The hybridoma cell strain is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody described in the seventh aspect.

In a ninth aspect, the present invention provides a Klebsiella pneumoniae polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K47 type Klebsiella pneumoniae, and the deposit number of the K47 type Klebsiella pneumoniae is GDMCC No: 62131. The sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 33, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 34.

Specifically, the complementarity determining region of the light chain variable region contains three sequences, as shown in SEQ ID: 35, SEQ ID: 36, and SEQ ID: 37, and the complementarity determining region of the heavy chain variable region contains three sequences, as shown in SEQ ID: 38, SEQ ID: 39, and SEQ ID: 40.

In a tenth aspect, the present invention provides a hybridoma cell strain, which is named K47-20C10-B8, and the deposit number of the hybridoma cell strain is: CCTCC NO: C2023313. The hybridoma cell strain is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody as described in the ninth aspect.

In the eleventh aspect, the present invention provides a Klebsiella pneumoniae polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae, and the deposit number of the K64 type Klebsiella pneumoniae is GDMCC No: 62132. The sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 41, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 42.

Specifically, the complementarity determining region of the light chain variable region contains three sequences, as shown in SEQ ID:43, SEQ ID:44, and SEQID:45, and the complementarity determining region of the heavy chain variable region contains three sequences, as shown in SEQ ID:46, SEQ ID:47, and SEQID:48.

In a twelfth aspect, the present invention provides a hybridoma cell strain, which is named K64-1, and the deposit number of the hybridoma cell strain is: CCTCC NO: C2023365. The hybridoma cell strain is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody as described in the eleventh aspect.

In a thirteenth aspect, the present invention provides a Klebsiella pneumoniae polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae, and the deposit number of the K64 type Klebsiella pneumoniae is GDMCC No: 62132. The sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 49, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 50.

Specifically, the complementarity determining region of the light chain variable region contains three sequences, as shown in SEQ ID:51, SEQ ID:52, and SEQID:53, and the complementarity determining region of the heavy chain variable region contains three sequences, as shown in SEQ ID:54, SEQ ID:55, and SEQID:56.

In a fourteenth aspect, the present invention provides a hybridoma cell strain, which is named K64-23, and the deposit number of the hybridoma cell strain is: CCTCC NO: C2023366. The hybridoma cell strain is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody as described in the thirteenth aspect.

In a fifteenth aspect, the present invention provides a Klebsiella pneumoniae polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae, and the preservation number of the K64 type Klebsiella pneumoniae is GDMCC No: 62132. The sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 57, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 58.

Specifically, the complementary determining region of the light chain variable region contains three sequences, as shown in SEQ ID:59, SEQ ID:60, and SEQID:61, and the complementary determining region of the heavy chain variable region contains three sequences, as shown in SEQ ID:62, SEQ ID:63, and SEQID:64.

In a sixteenth aspect, the present invention provides a hybridoma cell line, the hybridoma cell line is named K64-24, the deposit number of the hybridoma cell line is: CCTCC NO: C2023367, and the hybridoma cell line is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody as described in the fifteenth aspect. The present invention also provides a pharmaceutical composition, the pharmaceutical composition comprises any one of the aforementioned Klebsiella pneumoniae polysaccharide monoclonal antibodies, and the pharmaceutical composition further comprises any one or more combinations of pharmaceutically acceptable carriers, excipients or diluents.

The present invention also provides a kit for detecting K47 type Klebsiella pneumoniae, wherein the kit for detecting K47 type Klebsiella pneumoniae comprises the Klebsiella pneumoniae polysaccharide monoclonal antibody described in any one of the above, and the kit for detecting K47 type Klebsiella pneumoniae also comprises any one or more combinations of a positive control substance, a negative control substance, an antibody diluent, a color developing solution, a stop solution, a blocking solution or a washing solution.

The present invention also provides a kit for detecting K64 type Klebsiella pneumoniae, wherein the kit for detecting K64 type Klebsiella pneumoniae comprises any one of the aforementioned Klebsiella pneumoniae polysaccharide monoclonal antibodies, and the kit for detecting K64 type Klebsiella pneumoniae further comprises any one or more combinations of a positive control substance, a negative control substance, an antibody diluent, a color developing solution, a stop solution, a blocking solution, or a washing solution. The present invention also provides the use of any one of the aforementioned Klebsiella pneumoniae polysaccharide monoclonal antibodies in the preparation of a drug for treating a Klebsiella pneumoniae infection disease and/or a detection product.

Capsular polysaccharide is an important toxic factor of Klebsiella pneumoniae. The present invention improves immunogenicity by coupling capsular polysaccharide with carrier protein, and prepares K47 capsular polysaccharide-specific monoclonal antibody and K64 capsular polysaccharide-specific monoclonal antibody by hybridoma technology. The antibodies have the characteristics of good specificity, high affinity and high binding ability with Klebsiella pneumoniae capsular polysaccharide.

The beneficial effects of this application are:

The Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention can specifically bind to the capsular polysaccharide of Klebsiella K47 and Klebsiella K64, is beneficial to the typing of Klebsiella pneumoniae in clinic, and can prove that it has excellent effect in preventing and treating K47 and K64 Klebsiella pneumoniae infections in animal in vivo and in vitro tests, provides a way to solve the problems of Klebsiella pneumoniae infection and multidrug resistance, has significant significance for the prevention, diagnosis and treatment of Klebsiella pneumoniae infection, and has important value for the development of a new generation of drugs for Klebsiella pneumoniae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the titer level of the K47 type Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention detected by indirect ELISA in Example 2;

FIG2 is a flow cytometric analysis of the binding results of monoclonal antibody K47-20C10-B8 with Klebsiella pneumoniae encapsulated bacteria HVKP4 and non-encapsulated bacteria 19-249ΔwbaP (capsular polysaccharide expression gene knockout) in Example 4;

3 is a flow cytometric analysis of the binding results of monoclonal antibody K47-18G6-B2 with Klebsiella pneumoniae encapsulated bacteria HVKP4 and non-encapsulated bacteria 19-249ΔwbaP (capsular polysaccharide expression gene knockout) in Example 4;

FIG4 is a bactericidal curve of K47-5B9-F8 and K47-15D7-B10 against Klebsiella pneumoniae HVKP4 strain in vitro in Example 5;

FIG5 is a survival curve of the prevention test of the K47 type Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention in Example 6;

6 is a graph showing the titer level of the Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention detected by indirect ELISA in Example 8;

7 is a flow cytometry analysis of the binding of K64 monoclonal antibody, K47 monoclonal antibody and K64-type Klebsiella pneumoniae Y8 in Example 10;

FIG8 is a bactericidal curve of monoclonal antibodies K64-1, K64-23 and K64-24 against a highly virulent strain of Klebsiella pneumoniae (numbered Y8) in vitro in Example 11;

FIG. 9 is a survival curve of the K64-1 antibody preventive test in Example 12.

DETAILED DESCRIPTION

The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1: Preparation of monoclonal antibodies against Klebsiella pneumoniae K47 capsular polysaccharide

1) Establishment of cell line for monoclonal antibody against Klebsiella pneumoniae K47 capsular polysaccharide

The K47 type capsular polysaccharide protein conjugate is obtained by chemically coupling the capsular polysaccharide extracted from Klebsiella pneumoniae type K47 (GDMCC No: 62131) after fermentation with a carrier protein (diphtheria toxin-free variant CRM197). The method is a commonly used polysaccharide activation method, such as the cyanogen bromide method (reference US6375846B1), 1-cyano-4-dimethylamino arsenic tetrafluoroborate (CDAP) (EP0720485), and the periodic acid oxidation method (US4711779). The K47 capsular polysaccharide protein conjugate was added to Freund's adjuvant and SPF-grade BALB/c mice were immunized multiple times. When the immune titer was greater than 1:300,000, the spleen was taken and hybridoma technology was used to obtain 5 monoclonal cell lines, namely, hybridoma cell line K47-5B9-F8 (CCTCC NO: C2023314), hybridoma cell line K47-20C10-B8 (CCTCC NO: C2023313), hybridoma cell line K47-15D7-B10 (CCTCC NO: C2023312), hybridoma cell line K47-18G6-B2 (CCTCC NO: C2023311) and hybridoma cell line K47-6E2 (CCTCC NO: C2023296), and sequencing was used to verify their purity.

After sequencing, the sequence information of the five Klebsiella pneumoniae polysaccharide monoclonal antibodies is as follows (SEQ NO: 1 to SEQ NO: 40 in order):

K47-5B9-F8 - light chain variable region (VL) sequence:

MDFQVQIFSLLLISVTVIVSCGEIVLTQSPTTLAASPGEKITITCSASSSISSNYLHWYQQK

PGFSPKLLIYRTSNLASGLPARFSGSGSGTSYSLTIGTMEAEDVATYFCQQGGGIPRITFG

SGTKLEIK

K47-5B9-F8 - heavy chain variable region (VH) sequence:

MNFGLSWVFLVTLLNGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVR

QPPGKALEWLGFIRNKANGYTTEYTASVKGRFTISRDNSQSILYLQMNTLRAEDSATYY

CAREAYRFDGAMDYWGQGTSVTVSS

K47-5B9-F8 - light chain complementarity determining region 1 (LCDR1) sequence:

SASSSISSNYLH

K47-5B9-F8 - light chain complementarity determining region 2 (LCDR2) sequence:

RTSNLAS

K47-5B9-F8 - light chain complementarity determining region 3 (LCDR3) sequence:

QQGGGIPRIT

K47-5B9-F8 - heavy chain complementarity determining region 1 (HCDR1) sequence:

DYYMN

K47-5B9-F8 - heavy chain complementarity determining region 2 (HCDR2) sequence:

FIRNKANGYTTEYTASVKG

K47-5B9-F8 - heavy chain complementarity determining region 3 (HCDR3) sequence:

EAYRFDGAMDY K47-15D7-B10 - Light chain variable region (VL) sequence:

MDFQVQIFSLLLISVTVIVSNGEIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQ

KPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIPRITF

GSGTKLEIK

K47-15D7-B10 - Heavy chain variable region (VH) sequence:

MNFGLSWVFLVTLLNGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWFRQ

PPGKALEWLGFIRNKANGYTIEYTPSVQGRFTISRDNSQSILYLHMITLRTEDSATYYCA

REAYRFDGAMDYWGQGTSVTVSS

K47-15D7-B10 - light chain complementarity determining region 1 (LCDR1) sequence:

SASSSISSNYLH K47-15D7-B10 - Light chain complementarity determining region 2 (LCDR2) sequence:

RTSNLAS K47-15D7-B10 - light chain complementarity determining region 3 (LCDR3) sequence:

QQGSSIPRIT K47-15D7-B10 - Heavy chain complementarity determining region 1 (HCDR1) sequence:

DYYMN K47-15D7-B10 - heavy chain complementarity determining region 2 (HCDR2) sequence:

FIRNKANGYTIEYTPSVQG K47-15D7-B10 - heavy chain complementarity determining region 3 (HCDR3) sequence:

EAYRFDGAMDY K47-18G6-B2 - Light chain variable region (VL) sequence:

MSPAQFLVLLLFWIPASRGDVVLTQTPLSLPVSFGDQVSISCRSSQSLVNSYGITYLSWY

LHKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPW

TFGGGTKLEIK

K47-18G6-B2-heavy chain variable region (VH) sequence:

MEWTGIFILSVTAGVHSQVQLQQSGAELVRPGTSVKISCKASGYAFTNYWLGWIKQRP

GHGLEWIGDFYPGSGSTYYNEKFKGKATLTADKSSSTAYMQVSSLTSEDAAVYFCTRK

GFYGTSGRYFAYWGQGTTLTVSS

K47-18G6-B2 - Light chain complementarity determining region 1 (LCDR1) sequence:

RSSQSLVNSYGITYLS K47-18G6-B2 - Light chain complementarity determining region 2 (LCDR2) sequence:

GISNRFS K47-18G6-B2 - Light chain complementarity determining region 3 (LCDR3) sequence:

LQGTHQPWT K47-18G6-B2 - heavy chain complementarity determining region 1 (HCDR1) sequence:

NYWLG K47-18G6-B2 - heavy chain complementarity determining region 2 (HCDR2) sequence:

DFYPGSGSTYYNEKFKG K47-18G6-B2 - Heavy chain complementarity determining region 3 (HCDR3) sequence:

KGFYGTSGRYFAY K47-6E2-light chain variable region (VL) sequence:

MDFQVQIFSLLLISVTVMVSNGEIVLTQSPTTMAASPGEKITITCSASSSVRSNYLHWYQ

QKPGCSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSMPRI

TFGSGTKLEIN

K47-6E2-heavy chain variable region (VH) sequence:

MNFGLSWVFLVTLLNGIQCEVKLVESGGGLVLPGGSLRLSCTTSGFTFTDYYMNWVRQ

PPGKALEWLGFIRNKANGYTTDYNASVKGRFTISRDNSQSILYLQMNTLRAEDSASYY

CARESYRYDGAMDYWGQGTSVTVSS

K47-6E2-light chain complementarity determining region 1 (LCDR1) sequence:

SASSSVRSNYLH K47-6E2 - Light chain complementarity determining region 2 (LCDR2) sequence:

RTSNLAS K47-6E2 - light chain complementarity determining region 3 (LCDR3) sequence:

QQGSSMPRIT K47-6E2 - Heavy chain complementarity determining region 1 (HCDR1) sequence:

DYYMN K47-6E2 - heavy chain complementarity determining region 2 (HCDR2) sequence:

FIRNKANGYTTDYNASVKG K47-6E2 - heavy chain complementarity determining region 3 (HCDR3) sequence:

ESYRYDGAMDY

K47-20C10-B8 - light chain variable region (VL) sequence:

MESHSQVFIFLLFWIPVSRGDILLTQSPAILSVSPGERVSFSCRASQSIGRSIHWYQQRTN

GSPRLLIKYASESIYGIPSRFSGSGSGTDFTLSINSVESEDIAAYYCQQSYNWPRTFGGGT

KLEIK

K47-20C10-B8-heavy chain variable region (VH) sequence:

MNFGLSWVFLVALLNGVQCQVQLVETGGGLVRPGNSLNLSCITSGFTFSNYRLHWLRQ

PPGKGLEWLAVIAVKSDNFGAIYADSVKGRFTISRDDSRSSVYLQMNRLREEDTATYYC

VRAGVSFFDYWGQGTSLTVSS

K47-20C10-B8 - Light chain complementarity determining region 1 (LCDR1) sequence:

RASQSIGRSIH

K47-20C10-B8 - light chain complementarity determining region 2 (LCDR2) sequence:

YASESIY

K47-20C10-B8 - Light chain complementarity determining region 3 (LCDR3) sequence:

QQSYNWPRT

K47-20C10-B8 - heavy chain complementarity determining region 1 (HCDR1) sequence:

NYRLH

K47-20C10-B8 - heavy chain complementarity determining region 2 (HCDR2) sequence:

VIAVKSDNFGAIYADSVKG

K47-20C10-B8 - heavy chain complementarity determining region 3 (HCDR3) sequence:

AGVSFFDY

2) Preparation and purification of monoclonal antibodies against Klebsiella pneumoniae K47 polysaccharide in ascites

The monoclonal antibody cells obtained were expanded and cultured, and 0.5 mL of cells at a concentration of 1×10 ⁶ /mL were intraperitoneally injected into BALB/c mice sensitized with paraffin oil 7 days in advance. After 7-10 days, the abdomen of the mice was observed to have obvious bulges, and the ascites was collected for purification.

The mouse ascites was diluted with PBS, and 50% final concentration of ammonium sulfate was added for salt precipitation and then purified by Protein A affinity column chromatography and ion exchange chromatography to obtain a monoclonal antibody with a purity greater than 90%. After sterile filtration through a 0.22 μm filter membrane, it was stored in PBS at pH 7.0-7.4.

Example 2: Titer level detection of monoclonal antibodies to Klebsiella pneumoniae K47 capsular polysaccharide

After cell fusion, screening and several subcloning, 5 monoclonal antibody cell lines were obtained. The ELISA test results of the cell culture supernatant binding to K47 type capsular polysaccharide are shown in Table 1 below.

Table 1

Cell line number OD value K47-5B9-F8 2.335 K47-15D7-B10 1.826 K47-18G6-B2 2.158 K47-6E2 2.632 K47-20C10-B8 1.987

As shown in FIG1 , the titer levels of the Klebsiella pneumoniae polysaccharide monoclonal antibodies provided by the present invention were detected by indirect ELISA method, and it can be seen that the five monoclonal antibodies all have good binding affinity with K47 type capsular polysaccharide.

Example 3: ELISA detection of binding specificity of monoclonal antibodies to Klebsiella pneumoniae K47 capsular polysaccharide

The Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention reacts with K47, K64, K19, K1, K2, K38 and K57 capsular polysaccharides by indirect ELISA method to evaluate its specificity. The Klebsiella pneumoniae capsular polysaccharides of different serotypes were coated in an ELISA plate at 37°C for 3 hours, and after blocking with 1% BSA for 1 hour, an appropriate amount of K47 type Klebsiella pneumoniae polysaccharide monoclonal antibody was added to react with the coated polysaccharide, 50 μL per well, and incubated overnight. After washing the plate, the secondary antibody was diluted 1:30,000 and added to the ELISA plate, 100 μL per well, incubated for 2 hours, and then washed, and then 1 mg/mL of PNPP-Na colorimetric substrate was added, 100 μL per well, and after incubation for 2 hours, 3M NaOH was added to 50 μL wells to stop the reaction, and the OD405 value was read on the machine.

The results of the binding of the five Klebsiella pneumoniae polysaccharide monoclonal antibodies provided by the present invention to Klebsiella capsular polysaccharides of different serotypes are shown in Table 2 below:

Table 2

It can be seen from Table 2 above that the five Klebsiella pneumoniae polysaccharide monoclonal antibodies provided by the present invention are specific to K47 type capsular polysaccharide, and have no cross-reaction with other serotypes of Klebsiella capsular polysaccharides.

Example 4: Flow cytometry detection of binding between monoclonal antibody against Klebsiella pneumoniae K47 capsular polysaccharide and Klebsiella pneumoniae K47

The highly virulent Klebsiella pneumoniae strain HVKP4 and 19-249ΔwbaP strain (capsular polysaccharide expression gene knockout) were inoculated into 5 mL of LB medium and cultured at 37°C and 250 rpm until the OD value was 0.7. 0.2 mL of bacterial solution was centrifuged at 8000g for 10 min, the precipitate was collected, washed twice with PBS, and resuspended in 0.2 mL of PBS; then fixed with 4% paraformaldehyde for 10 min, blocked with 2% BSA for 1 h after washing, and then incubated with K47 type capsular polysaccharide monoclonal antibody overnight, and after washing, anti-mouse fluorescent antibody (brand-CST, item number-#4410S) was added and incubated in the dark for 1 h, washed with PBS, resuspended in PBS and detected by flow cytometry.

The test results are shown in Figure 2, which is a flow cytometry analysis of the binding peaks of monoclonal antibody K47-20C10-B8 with Klebsiella pneumoniae encapsulated bacteria HVKP4 and non-encapsulated bacteria 19-249ΔwbaP (capsular polysaccharide expression gene knockout), and Figure 3 is a flow cytometry analysis of the binding peaks of monoclonal antibody K47-18G6-B2 with Klebsiella pneumoniae HVKP4 and bacteria 19-249ΔwbaP (capsular polysaccharide expression gene knockout).

As shown in Figure 2, monoclonal antibody K47-20C10-B8 can bind to the highly virulent strain HVKP4 of Klebsiella pneumoniae, but not to Klebsiella pneumoniae 19-249ΔwbaP (capsular polysaccharide expression gene knockout). It can be seen that monoclonal antibody K47-20C10-B8 can be used for the diagnosis and typing of clinical strains.

Referring to Figure 3, monoclonal antibody K47-18G6-B2 was tested for binding to Klebsiella pneumoniae HVKP4. The results showed that monoclonal antibody K47-18G6-B2 could bind to the encapsulated strains but not to Klebsiella pneumoniae 19-249ΔwbaP (capsular polysaccharide expression gene knockout). It can be seen that monoclonal antibody K47-18G6-B2 can be used for the diagnosis and typing of clinical strains.

Example 5: Bactericidal test of monoclonal antibody against Klebsiella pneumoniae K47 capsular polysaccharide

This example shows the opsonization and phagocytosis bactericidal test of the K47 type Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention, specifically: the carbapenem-resistant Klebsiella pneumoniae HVKP4 strain is diluted to 10 ⁵ CFU/mL and added to the 96-well cell working plate at 10 μL/well. The Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention is gradiently diluted and added to the above-mentioned cell working plate at 20 μL/well, and the bacteria and the antibody are incubated at 700 rpm/min for 30 minutes, and the HL-60 cells differentiated by DMF are washed with HBSS buffer and adjusted to a concentration of 1×10 ⁷ cells/mL, and then 1×10 ⁷ cells/mL of cell fluid is mixed with the diluted complement at a volume ratio of 1:4, and the mixed solution is added to the 96-well cell working plate at 50 μL/well. The 96-well cell working plate is placed on a mixer, placed in a CO ₂ incubator with 5% CO ₂ and a temperature of 37°C, and shaken for 45 minutes. After terminating opsonophagocytosis, the samples were spotted onto blood plates and incubated overnight in a CO ₂ incubator. The bactericidal rate of each diluted antibody was calculated.

As shown in Figure 4, the sterilization curves of K47-5B9-F8 and K47-15D7-B10 of the K47 type Klebsiella pneumoniae polysaccharide monoclonal antibodies provided by the present invention against the Klebsiella pneumoniae HVKP4 strain in vitro, according to Figure 4, it can be seen that K47-5B9-F8 and K47-15D7-B10 have good sterilization effects on the highly toxic strain HVKP4 in vitro, and the highest sterilization rates reach 91.5% and 95.5%, respectively. There is a significant difference compared with the negative control (K64 type capsular polysaccharide monoclonal antibody).

Example 6: Preventive test of monoclonal antibodies against Klebsiella pneumoniae K47 capsular polysaccharide

This example shows the protective test of the K47 type Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention, specifically: 5 kinds of Klebsiella pneumoniae polysaccharide monoclonal antibodies were administered by intraperitoneal injection, 5 mice in each group, and each mouse was given 0.25 mg, PBS was used as a negative control, and 4 hours later, Balb/c mice (n=5) were infected intraperitoneally with the K47 type Klebsiella pneumoniae highly virulent strain HVKP4 at a bacterial amount of 8.5×10 ⁵ CFU, and the survival rate of the mice was observed for 10 days after infection, and a survival curve was drawn.

As shown in FIG5 , it is the survival curve of the preventive test of the K47 Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention. It can be seen that 4 hours after administration of the five K47 capsular polysaccharide monoclonal antibodies and the control PBS, the Klebsiella pneumoniae K47 bacteria (strain number HVKP4) were challenged with 8.5×10 ⁵ CFU. The survival rate of the control group was 20%, and the survival rates of the five monoclonal antibody groups were all 100%, with statistically significant differences, p<0.0001.

Example 7: Preparation of monoclonal antibodies against Klebsiella pneumoniae K64 capsular polysaccharide

1) Establishment of cell line for monoclonal antibody against Klebsiella pneumoniae K64 capsular polysaccharide

The K64 type capsular polysaccharide protein conjugate is obtained by chemically coupling the capsular polysaccharide extracted from Klebsiella pneumoniae K64 type (GDMCC No: 62132) after fermentation with a carrier protein (diphtheria toxin non-toxic variant CRM197). The method is a commonly used polysaccharide activation method, such as cyanogen bromide method (reference US6375846B1), 1-cyano-4-dimethylamino arsenic tetrafluoroborate (CDAP) (EP0720485), and periodic acid oxidation method (US4711779). The K64-type polysaccharide-protein conjugate was added to Freund's adjuvant and SPF-grade BALB/c mice were immunized multiple times. When the immune titer was greater than 1:300,000, the spleen was taken and hybridoma technology was used to obtain three monoclonal cell lines, namely, hybridoma cell line K64-1 (CCTCC NO: C2023365), hybridoma cell line K64-23 (CCTCC NO: C2023366) and hybridoma cell line K64-24 (CCTCC NO: C2023367), which were verified to be pure by sequencing.

After sequencing, the sequence information of the three Klebsiella pneumoniae polysaccharide monoclonal antibodies are as follows (SEQ NO: 41 to SEQ NO: 64, in order):

K64-1 light chain variable region (VL) sequence (starting from FR-L1):

QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPA

RFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPWTFGGGTKLEIK K64-1 heavy chain variable region (VH) sequence (starting from FR-H1):

EVKLVESGGGLVKPGGSLKLSCAGSGFTFSSYGMSWVRQTPDKRLEWVATISGGGSYT

YYSDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARNYGYDGHYYAMDYWGQ

GTSVTVSS

Complementarity determining region 1 (LCDR1) sequence of K64-1 light chain:

SASSSISYMH K64-1 light chain complementarity determining region 2 (LCDR2) sequence:

DTSKLAS K64-1 light chain complementarity determining region 3 (LCDR3) sequence:

HQRSSYPWT K64-1 heavy chain complementarity determining region 1 (HCDR1) sequence:

Complementarity determining region 2 (HCDR2) sequence of SYGMS K64-1 heavy chain:

TISGGGSYTYYSDSVKG K64-1 heavy chain complementarity determining region 3 (HCDR3) sequence:

NYGYDGHYYAMDY K64-23 light chain variable region (VL) sequence (starting from FR-L1):

QIVLTQSPAIMSASPGEKVTMTCSATSIVNYMHWYQQKSGTSPKRWIYDTSKLASGVPA

RFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPRT K64-23 heavy chain variable region (VH) sequence (starting from FR-H1):

QVQLQQPWSELVRPGASVKLPCKASGYTFTSYWMNWVKQRPEQGLEWIGRIDPYDG

GTHYSQNFMDRAILTVDRSSNTAYMQLNSLTSEDSAVYYCARGYANGAFVYWGQGTL

VTVSA

K64-23 light chain complementarity determining region 1 (LCDR1) sequence:

Complementarity determining region 2 (LCDR2) sequence of the light chain of SATSIVNYMH K64-23:

DTSKLAS K64-23 light chain complementarity determining region 3 (LCDR3) sequence:

QQWSSNPRT

Complementarity determining region 1 (HCDR1) sequence of K64-23 heavy chain:

SYWMN

Complementarity determining region 2 (HCDR2) sequence of K64-23 heavy chain:

RIDPYDGGTHYSQNFMD

Complementarity determining region 3 (HCDR3) sequence of K64-23 heavy chain:

GYANGAFVY

K64-24 light chain variable region (VL) sequence (starting from FR-L1):

QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQRPGSSPKPWIYRTSNLASGVPAR

FSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPLTFGAGTKLELK

K64-24 heavy chain variable region (VH) sequence (starting from FR-H1):

EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAYMDWVRQSPEKGLEWVAEIRSKANN

HATLYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCTPYYYGSSQFAYWGQGTL

VTVSA

Complementarity determining region 1 (LCDR1) sequence of K64-24 light chain:

SASSSVSYMY

Complementarity determining region 2 (LCDR2) sequence of K64-24 light chain:

RTSNLAS

Complementarity determining region 3 (LCDR3) sequence of K64-24 light chain:

QQYHSYPLT

Complementarity determining region 1 (HCDR1) sequence of K64-24 heavy chain:

DAYMD

Complementarity determining region 2 (HCDR2) sequence of K64-24 heavy chain:

EIRSKANNHATLYAESVKG

Complementarity determining region 3 (HCDR3) sequence of K64-24 heavy chain:

YYYGSSQFAY

2) Preparation and purification of monoclonal antibodies against Klebsiella pneumoniae K64 capsular polysaccharide in ascites

The monoclonal antibody cells obtained were expanded and cultured, and 0.5 mL of cells at a concentration of 1×10 ⁶ /mL were intraperitoneally injected into BALB/c mice sensitized with paraffin oil 7 days in advance. After 7-10 days, the abdomen of the mice was observed to have obvious bulges, and the ascites was collected for purification.

The mouse ascites was diluted with PBS, and 50% final concentration of ammonium sulfate was added for salt precipitation and then purified by Protein A affinity column chromatography and ion exchange chromatography to obtain a monoclonal antibody with a purity greater than 95%. After sterile filtration through a 0.22 μm filter membrane, it was stored in PBS at pH 7.0-7.4.

Example 8: Titer level detection of monoclonal antibodies to Klebsiella pneumoniae K64 capsular polysaccharide

After cell fusion, screening and several subclones, three monoclonal antibody cell lines were obtained. The results of ELISA test on the binding of cell culture supernatants to K64-type capsular polysaccharide are shown in Table 3 below.

Table 3

Cell line number OD value K64-1 3.816 K64-23 1.315 K64-24 2.011

As shown in FIG6 , the titer level of the Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention was detected by indirect ELISA method, and it can be seen that the supernatants of the three cell strains all have good binding affinity with the K64-type capsular polysaccharide.

Example 9: ELISA detection of binding specificity of monoclonal antibodies to Klebsiella pneumoniae K64 capsular polysaccharide

The Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention reacts with K47, K64, K19, K1, K2, K38 and K57 capsular polysaccharides by indirect ELISA method to evaluate its specificity. The Klebsiella pneumoniae capsular polysaccharides of different serotypes were coated in an ELISA plate at 37°C for 3 hours, and after blocking with 1% BSA for 1 hour, an appropriate amount of K64 Klebsiella pneumoniae polysaccharide monoclonal antibody was added to react with the coated polysaccharide, 50 μL per well, and incubated overnight. After washing the plate, the AP-labeled goat anti-mouse secondary antibody was diluted 1:30,000 and added to the ELISA plate, 100 μL per well, incubated for 2 hours, and then washed, and then 1 mg/mL of PNPP-Na color substrate was added, 100 μL per well, and after incubation for 2 hours, 3M NaOH was added to 50 μL wells to stop the reaction, and the OD405 value was read on the machine.

The results of the three K64 Klebsiella pneumoniae polysaccharide monoclonal antibodies provided by the present invention binding to Klebsiella capsular polysaccharides of different serotypes are shown in Table 4 below:

Table 4

It can be seen from Table 4 above that the three K64-type Klebsiella pneumoniae polysaccharide monoclonal antibodies provided by the present invention are specific to K64-type capsular polysaccharide and have no cross-reaction with other serotypes of Klebsiella capsular polysaccharide.

Example 10: Flow cytometry detection of binding between monoclonal antibody against Klebsiella pneumoniae K64 capsular polysaccharide and Klebsiella pneumoniae K64

K64-Serotype highly virulent Klebsiella pneumoniae Y8 (Li, J.; Sheng, Y.; Ma, R.; Xu, M.; Liu, F.; Qin, R.; Zhu, M.; Zhu, X.; He, P. Identification of a Depolymerase Specific for K64-Serotype Klebsiella pneumoniae: Potential Applications in Capsular Typing and Treatment. Antibiotics 2021, 10, 144.) was inoculated into 5 ml LB medium and cultured at 37°C and 250 rpm until the OD value was 0.7. 0.2 mL of the bacterial solution was taken and centrifuged at 8000 g for 10 min. The precipitate was collected, washed twice with PBS, and resuspended in 0.2 mL PBS; then fixed with 4% paraformaldehyde for 10 minutes, blocked with 2% BSA for 1 hour after washing, and then incubated with K64 capsular polysaccharide monoclonal antibody overnight, added with anti-mouse fluorescent antibody (brand-CST, item number-#4410S) after washing, incubated in the dark for 1 hour, washed with PBS, resuspended in PBS and detected by flow cytometry.

After culturing, the highly virulent Klebsiella pneumoniae strain Y8 was combined with K64-type monoclonal antibodies, and K47-20C10-B8 monoclonal antibody was used as a negative control. The binding ability was analyzed by flow cytometry. The test results are shown in Figure 7. The three K64-type monoclonal antibodies and K47-20C10-B8 monoclonal antibodies were respectively combined with the K64-type highly virulent Klebsiella pneumoniae strain Y8 for detection. The results showed that the three K64-type monoclonal antibodies could bind to Y8, while K47-20C10-B8 could not bind to Y8, indicating that the three K64-type capsular polysaccharide monoclonal antibodies can be used in the diagnosis and typing of clinical strains.

Example 11: Bactericidal test of monoclonal antibody against Klebsiella pneumoniae K64 capsular polysaccharide

This example shows the opsonization phagocytosis bactericidal test of the K64 type Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention, specifically: the K64 type highly virulent Klebsiella pneumoniae Y8 strain is diluted to 10 ⁵ CFU/ml and added to the 96-well cell working plate at 10 μL/well. The K64 type capsular polysaccharide monoclonal antibody is gradiently diluted and added to the above-mentioned cell working plate at 20 μL/well, and the bacteria and the antibody are incubated at 700 rpm/min for 30 minutes, and the HL-60 cells differentiated by DMF are washed with HBSS buffer and adjusted to a concentration of 1×10 ⁷ cells/ml, and then the 1×107 cells/ml cell fluid is mixed with the diluted complement at a volume ratio of 1:4, and the mixed solution is added to the 96-well cell working plate at 50 μL. The 96-well cell working plate is placed on a mixer, placed in a CO ₂ incubator with 5% CO ₂ and a temperature of 37°C, and shaken for 45 minutes. After terminating opsonophagocytosis, the samples were spotted onto blood plates and incubated overnight in a CO ₂ incubator. The bactericidal rate of each diluted antibody was calculated.

As shown in Figure 8, K64-type capsular polysaccharide monoclonal antibodies K64-1, K64-23, and K64-24 had good bactericidal effects on the highly virulent strain Y8 in vitro, with the highest bactericidal rates reaching 83.3% and 86.7%, respectively, which were significantly different from the negative control (K47-type capsular polysaccharide monoclonal antibody).

Example 12: Prophylactic test of monoclonal antibodies against Klebsiella pneumoniae K64 capsular polysaccharide

This example shows the protective test of the Klebsiella pneumoniae polysaccharide monoclonal antibody provided by the present invention, specifically: three types of K64 type Klebsiella pneumoniae polysaccharide monoclonal antibodies were administered by intraperitoneal injection, with 6 mice in each group, and 0.025 mg was given to each mouse, PBS was used as a negative control, and 4 hours later, Balb/c mice (n=6) were infected intraperitoneally with the highly virulent Klebsiella pneumoniae strain Y8 at a bacterial amount of 7.38×10 ⁶ CFU, and the survival rate of the mice was observed for 10 days after infection, and a survival curve was drawn.

As shown in FIG9 , it can be seen that 4 hours after administration of the monoclonal antibody K64-1 against K64 capsular polysaccharide and the control PBS, the survival rate of the control group was 0%, and the survival rate of the monoclonal antibody group was ¹⁰⁰ %, with a statistically significant difference (p<0.05).

In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims (28)

1. A Klebsiella pneumoniae polysaccharide monoclonal antibody, characterized in that the Klebsiella pneumoniae is K47 type Klebsiella pneumoniae, the deposit number of the K47 type Klebsiella pneumoniae is GDMCC No: 62131, the sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 2. 2. The Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 1, characterized in that the complementary determining region of the light chain variable region comprises three sequences as shown in SEQ ID: 3, SEQ ID: 4, and SEQ ID: 5, and the complementary determining region of the heavy chain variable region comprises three sequences as shown in SEQ ID: 6, SEQ ID: 7, and SEQ ID: 8. 3. A hybridoma cell strain, characterized in that the hybridoma cell strain is named K47-5B9-F8, the deposit number of the hybridoma cell strain is: CCTCC NO: C2023314, and the hybridoma cell strain is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody as claimed in claim 1. 4. A Klebsiella pneumoniae polysaccharide monoclonal antibody, characterized in that the Klebsiella pneumoniae is K47 type Klebsiella pneumoniae, the deposit number of the K47 type Klebsiella pneumoniae is GDMCC No: 62131, the sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 9, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 10. 5. The Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 4, characterized in that the complementary determining region of the light chain variable region comprises three sequences, as shown in SEQ ID: 11, SEQ ID: 12, and SEQ ID: 13, and the complementary determining region of the heavy chain variable region comprises three sequences, as shown in SEQ ID: 14, SEQ ID: 15, and SEQ ID: 16. 6. A hybridoma cell line, characterized in that the hybridoma cell line is named K47-15D7-B10, the deposit number of the hybridoma cell line is: CCTCC NO: C2023312, and the hybridoma cell line is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody as claimed in claim 4. 7. A Klebsiella pneumoniae polysaccharide monoclonal antibody, characterized in that the Klebsiella pneumoniae is K47 type Klebsiella pneumoniae, the deposit number of K47 type Klebsiella pneumoniae is GDMCC No: 62131, the sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 17, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 18. 8. The Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 7, characterized in that the complementary determining region of the light chain variable region comprises three sequences as shown in SEQ ID: 19, SEQ ID: 20, and SEQ ID: 21, and the complementary determining region of the heavy chain variable region comprises three sequences as shown in SEQ ID: 22, SEQ ID: 23, and SEQ ID: 24. 9. A hybridoma cell line, characterized in that the hybridoma cell line is named K47-18G6-B2, the deposit number of the hybridoma cell line is: CCTCC NO: C2023311, and the hybridoma cell line is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 7. 10. A Klebsiella pneumoniae polysaccharide monoclonal antibody, characterized in that the Klebsiella pneumoniae is K47 type Klebsiella pneumoniae, the deposit number of K47 type Klebsiella pneumoniae is GDMCC No: 62131, the sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 25, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 26. 11. The Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 10, characterized in that the complementary determining region of the light chain variable region comprises three sequences, as shown in SEQ ID: 27, SEQ ID: 28 and SEQ ID: 29, and the complementary determining region of the heavy chain variable region comprises three sequences, as shown in SEQ ID: 30, SEQ ID: 31 and SEQ ID: 32. 12. A hybridoma cell line, characterized in that the hybridoma cell line is named K47-6E2, the deposit number of the hybridoma cell line is: CCTCC NO: C2023296, and the hybridoma cell line is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 10. 13. A Klebsiella pneumoniae polysaccharide monoclonal antibody, characterized in that the Klebsiella pneumoniae is K47 type Klebsiella pneumoniae, the deposit number of K47 type Klebsiella pneumoniae is GDMCC No: 62131, the sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 33, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 34. 14. The Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 13, characterized in that the complementary determining region of the light chain variable region comprises three sequences as shown in SEQ ID: 35, SEQ ID: 36, and SEQ ID: 37, and the complementary determining region of the heavy chain variable region comprises three sequences as shown in SEQ ID: 38, SEQ ID: 39, and SEQ ID: 40. 15. A hybridoma cell line, characterized in that the hybridoma cell line is named K47-20C10-B8, the deposit number of the hybridoma cell line is: CCTCC NO: C2023313, and the hybridoma cell line is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 13. 16. A Klebsiella pneumoniae polysaccharide monoclonal antibody, characterized in that the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae, the deposit number of K64 type Klebsiella pneumoniae is GDMCC No: 62132, the sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 41, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 42. 17. The Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 16, characterized in that the complementary determining region of the light chain variable region comprises three sequences as shown in SEQ ID:43, SEQ ID:44, and SEQ ID:45, and the complementary determining region of the heavy chain variable region comprises three sequences as shown in SEQ ID:46, SEQ ID:47, and SEQ ID:48. 18. A hybridoma cell line, characterized in that the hybridoma cell line is named K64-1, the deposit number of the hybridoma cell line is: CCTCC NO: C2023365, and the hybridoma cell line is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 16. 19. A Klebsiella pneumoniae polysaccharide monoclonal antibody, characterized in that the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae, the deposit number of K64 type Klebsiella pneumoniae is GDMCC No: 62132, the sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 49, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 50. 20. The Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 19, characterized in that the complementary determining region of the light chain variable region comprises three sequences, as shown in SEQ ID: 51, SEQ ID: 52, and SEQ ID: 53, and the complementary determining region of the heavy chain variable region comprises three sequences, as shown in SEQ ID: 54, SEQ ID: 55, and SEQ ID: 56. 21. A hybridoma cell line, characterized in that the hybridoma cell line is named K64-23, the deposit number of the hybridoma cell line is: CCTCC NO: C2023366, and the hybridoma cell line is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 19. 22. A Klebsiella pneumoniae polysaccharide monoclonal antibody, characterized in that the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae, the deposit number of K64 type Klebsiella pneumoniae is GDMCC No: 62132, the sequence of the Klebsiella pneumoniae polysaccharide monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ NO: 57, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO: 58. 23. The Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 22, characterized in that the complementary determining region of the light chain variable region comprises three sequences as shown in SEQ ID: 59, SEQ ID: 60, and SEQ ID: 61, and the complementary determining region of the heavy chain variable region comprises three sequences as shown in SEQ ID: 62, SEQ ID: 63, and SEQ ID: 64. 24. A hybridoma cell line, characterized in that the hybridoma cell line is named K64-24, the deposit number of the hybridoma cell line is: CCTCC NO: C2023367, and the hybridoma cell line is used to prepare the Klebsiella pneumoniae polysaccharide monoclonal antibody according to claim 22. 25. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the Klebsiella pneumoniae polysaccharide monoclonal antibody according to any one of claims 1, 4, 7, 10, 13, 16, 19 and 22, and further comprises any one or more combinations of pharmaceutically acceptable carriers, excipients or diluents. 26. A kit for detecting K47 type Klebsiella pneumoniae, characterized in that the kit for detecting K47 type Klebsiella pneumoniae comprises the Klebsiella pneumoniae polysaccharide monoclonal antibody described in any one of claims 1, 4, 7, 10 and 13, and the kit for detecting K47 type Klebsiella pneumoniae also comprises any one or more combinations of a positive control, a negative control, an antibody diluent, a color developing solution, a stop solution, a blocking solution or a washing solution. 27. A kit for detecting K64 type Klebsiella pneumoniae, characterized in that the kit for detecting K64 type Klebsiella pneumoniae comprises the Klebsiella pneumoniae polysaccharide monoclonal antibody according to any one of claims 16, 19 and 22, and the kit for detecting K64 type Klebsiella pneumoniae also comprises any one or more combinations of a positive control, a negative control, an antibody diluent, a color developing solution, a stop solution, a blocking solution or a washing solution. 28. Use of a monoclonal antibody against Klebsiella pneumoniae polysaccharide as claimed in any one of claims 1, 4, 7, 10, 13, 16, 19 and 22 in the preparation of a drug for treating a Klebsiella pneumoniae infection and/or a detection product.