Indirect ELISA method for detecting Xinxiang babesiosis of sheep

Abstract

The invention discloses an ELISA diagnostic kit for detecting Xinxiang babesiosis of sheep. The kit comprises a recombinant RAP-1 protein, which is prepared by an escherichia coli expression system and is taken as the antigen for preventing specific antibody of Xinjiang babeisa in sheep.

Description

Technical field

[0001] The invention relates to an ELISA diagnostic kit for detecting Babesia Xinjiang in sheep, in particular to a recombinant RAP-1 protein prepared by an E. coli expression system as an antigen for detecting specific antibodies against Babesia Xinjiang in serum. And established an indirect ELISA method. Background technique

[0002] Ovine Babesiosis (ovine Babesiosis) is transmitted by vector ticks by Babesia ovis (Babesia ovis), Babesia ovis (B.motasi) and Babesiosis rough (B). .crassa) is the general term for diseases caused by parasitic parasitism in the red blood cells of sheep and goats. It is a blood protozoan mainly distributed in tropical and subtropical regions. The clinical response is usually characterized by high fever, hemolytic anemia, jaundice, and hemoglobinuria, which can cause death in severe cases. The disease is serious and can cause death in severe cases and cause heavy economic losses to the sheep industry.

[0003] Babesia sheep reprodu...

Examples

Embodiment 1

[0035] Example 1 Cloning of RAP-1 gene

[0036] 1. PCR amplification: Using Babesia Xinjiang unspecified cDNA as template, upstream primer (rRAP-1aα-CT-F): 5'-CGCATATGATCGCCATTCCAACAAAAGA-3' (SEQ ID NO.1); downstream primer (rRAP-1aα) -R): 5'-GCCAAGCTTTTCTTGAGATACCTCATCCT-3' (SEQ ID NO. 1) for PCR amplification. The reaction system is 50μL, namely: 10× buffer 5μL, dNTPs (2.5mmol / L) 4μL, upstream primer 1μL, downstream primer 1μL, template (cDNA) 2μL, sterilized water 36.5μL, Taq enzyme (Takara, RR001A) ( 5U / μL) 0.25μL.

[0037] After centrifugation and mixing, it was placed in a PCR automatic amplification instrument, and pre-denatured at 94°C for 5 minutes, and the following cycles were carried out: 94°C for 1 minute, 55°C for 30s, 72°C for 45s, after 35 cycles, 72°C for 10 minutes. Load 5 μL of the PCR product and perform electrophoresis in an agarose gel (80-100V). If the size of the amplified product fragment is consistent with the expected target fragment size, it can be use...

Embodiment 2

[0075] Example 2 Construction of recombinant expression vector

[0076] Enzyme digestion and recovery of the target fragment and pET30a-RAP vector:

[0077] The sequenced pGEM-rap-1 plasmid and pET30a plasmid were digested with Nde I and Hind Ⅲ respectively.

[0078] The digestion system (50μL) is as follows, the target gene tube is 10×buffer K 5μL, BSA 1μL, Hind Ⅲ 2.5μL, Nde I 2.5μL, H 2 O 24μL, pGEM-rap-1 plasmid 15μL. The carrier tube is 10×buffer K 5μL, BSA 1μL, Nde I 2.5μL, Hind III 2.5μL, H 2 O 24μL, pET30a vector 15μL.

[0079] ② After the enzyme digestion mixture is centrifuged and mixed, it is digested in a 37℃ water bath for 3 hours. ③Take 3uL digestion mixture for electrophoresis observation. After digestion is complete, incubate the endonuclease in a water bath at 65°C for 15min. Use 1% agarose gel electrophoresis of the recombinant plasmid and vector digestion products to cut the desired bands. , Use Agarose Gel DNA Extraction Kit to recover and purify the target fragme...

Embodiment 3

[0087] Example 3 Induced expression of recombinant expression plasmid

[0088] 5 μL of the recombinant bacteria after sequence analysis was inoculated into 3 mL of LB culture medium (containing 50 μg / mL carbenicillin), and shaken overnight at 37°C. Take 200μL of the overnight culture and inoculate it in 20mL of LB broth (containing 50μg / mL carbenicillin), shake vigorously to OD 600 When it was 0.6-1.0, IPTG was added to a final concentration of 1mmol / L, and expression was induced at 37°C. The bacterial solution was collected at the 4th hour, and analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting to detect the expression product. At the same time, the uninduced bacterial liquid was used as a negative control.

Technical field

[0001] The invention relates to an ELISA diagnostic kit for detecting Babesia Xinjiang in sheep, in particular to a recombinant RAP-1 protein prepared by an E. coli expression system as an antigen for detecting specific antibodies against Babesia Xinjiang in serum. And established an indirect ELISA method. Background technique

[0002] Ovine Babesiosis (ovine Babesiosis) is transmitted by vector ticks by Babesia ovis (Babesia ovis), Babesia ovis (B.motasi) and Babesiosis rough (B). .crassa) is the general term for diseases caused by parasitic parasitism in the red blood cells of sheep and goats. It is a blood protozoan mainly distributed in tropical and subtropical regions. The clinical response is usually characterized by high fever, hemolytic anemia, jaundice, and hemoglobinuria, which can cause death in severe cases. The disease is serious and can cause death in severe cases and cause heavy economic losses to the sheep industry.

[0003] Babesia sheep reprodu...

Examples

Embodiment 1

[0035] Example 1 Cloning of RAP-1 gene

[0036] 1. PCR amplification: Using Babesia Xinjiang unspecified cDNA as template, upstream primer (rRAP-1aα-CT-F): 5'-CGCATATGATCGCCATTCCAACAAAAGA-3' (SEQ ID NO.1); downstream primer (rRAP-1aα) -R): 5'-GCCAAGCTTTTCTTGAGATACCTCATCCT-3' (SEQ ID NO. 1) for PCR amplification. The reaction system is 50μL, namely: 10× buffer 5μL, dNTPs (2.5mmol / L) 4μL, upstream primer 1μL, downstream primer 1μL, template (cDNA) 2μL, sterilized water 36.5μL, Taq enzyme (Takara, RR001A) ( 5U / μL) 0.25μL.

[0037] After centrifugation and mixing, it was placed in a PCR automatic amplification instrument, and pre-denatured at 94°C for 5 minutes, and the following cycles were carried out: 94°C for 1 minute, 55°C for 30s, 72°C for 45s, after 35 cycles, 72°C for 10 minutes. Load 5 μL of the PCR product and perform electrophoresis in an agarose gel (80-100V). If the size of the amplified product fragment is consistent with the expected target fragment size, it can be use...

Embodiment 2

[0075] Example 2 Construction of recombinant expression vector

[0076] Enzyme digestion and recovery of the target fragment and pET30a-RAP vector:

[0077] The sequenced pGEM-rap-1 plasmid and pET30a plasmid were digested with Nde I and Hind Ⅲ respectively.

[0078] The digestion system (50μL) is as follows, the target gene tube is 10×buffer K 5μL, BSA 1μL, Hind Ⅲ 2.5μL, Nde I 2.5μL, H 2 O 24μL, pGEM-rap-1 plasmid 15μL. The carrier tube is 10×buffer K 5μL, BSA 1μL, Nde I 2.5μL, Hind III 2.5μL, H 2 O 24μL, pET30a vector 15μL.

[0079] ② After the enzyme digestion mixture is centrifuged and mixed, it is digested in a 37℃ water bath for 3 hours. ③Take 3uL digestion mixture for electrophoresis observation. After digestion is complete, incubate the endonuclease in a water bath at 65°C for 15min. Use 1% agarose gel electrophoresis of the recombinant plasmid and vector digestion products to cut the desired bands. , Use Agarose Gel DNA Extraction Kit to recover and purify the target fragme...

Embodiment 3

[0087] Example 3 Induced expression of recombinant expression plasmid

[0088] 5 μL of the recombinant bacteria after sequence analysis was inoculated into 3 mL of LB culture medium (containing 50 μg / mL carbenicillin), and shaken overnight at 37°C. Take 200μL of the overnight culture and inoculate it in 20mL of LB broth (containing 50μg / mL carbenicillin), shake vigorously to OD 600 When it was 0.6-1.0, IPTG was added to a final concentration of 1mmol / L, and expression was induced at 37°C. The bacterial solution was collected at the 4th hour, and analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting to detect the expression product. At the same time, the uninduced bacterial liquid was used as a negative control.

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