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Preparation method of HRPII protein monoclonal antibody of plasmodium falciparum

A technology of Plasmodium falciparum and monoclonal antibody, which is applied in the fields of botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of false positive test results, difficulty in preparing monoclonal antibodies, and difficulty in expression, etc. To achieve the effect of high specificity, enhanced stimulation, and improved expression level

Active Publication Date: 2010-03-03
杭州新脉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The immunogen used in the preparation of conventional HRPII monoclonal antibodies is the complete HRPII protein expressed by genetic engineering technology. Due to the base codon, it is difficult to express the protein in E. coli, and the expression product is mainly inclusion bodies, which makes monoclonal The preparation of resistance is very difficult
In addition, due to the homology of the antigenic epitope, the monoclonal antibody prepared using the intact HRPII protein may also recognize other proteins, such as rheumatoid factor, resulting in false positive test results

Method used

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  • Preparation method of HRPII protein monoclonal antibody of plasmodium falciparum

Examples

Experimental program
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Effect test

Embodiment 1

[0006] Example 1: Selection of dominant antigenic epitopes of Plasmodium falciparum HRPII.

[0007] Using the Plasmodium falciparum HRPII protein as the target antigen, the hydrophilicity and antigenicity of its amino acid sequence were analyzed using the biological software DNAssist2.0, and two dominant antigenic epitopes, A and B, were selected. The results of sequence comparison showed that the selected two dominant antigenic epitopes A and B had high sequence specificity and no obvious homology with other protein sequences.

[0008] A: LeuAsnLeuAsnLysArgLeuLeuHisGluThrGlnAlaHisValAspAspAlaHisHisAlaHisHisValAlaAspAlaHisHisAlaHis

[0009] B: AspAlaArgHisAlaThrAspAlaHisHisAlaAlaAspAlaHisHisAlaThrAspAlaHis.

Embodiment 2

[0010] Example 2: Concatenation of HRPII dominant epitopes of Plasmodium falciparum.

[0011] In order to enhance the stimulation of the target antigen epitope on the mouse immune system, the two dominant antigen epitope sequences of Plasmodium falciparum HRPII protein A and B were respectively repeated and then connected by a flexible fragment (four consecutive glycines) to obtain recombinant protein C amino acids sequence, the specific sequence is as follows:

[0012] LeuAsnLeuAsnLysArgLeuLeuHisGluThrGlnAlaHisValAspAspAlaHisHisAlaHisHisValAlaAspAlaHisHisAlaHisLeuAsnLeuAsnLysArgLeuLeuHisGluThrGlnAlaHisValAspAspAlaHisHisAlaHisHisValAlaAspAlaHisHisAlaHisGlyGlyGlyGlyAspAlaArgHisAlaThrAspAlaHisHisAlaAlaAspAlaHisHisAlaThrAspAlaHisAspAlaArgHisAlaThrAspAlaHisHisAlaAlaAspAlaHisHisAlaThrAspAlaHis。

Embodiment 3

[0013] Example 3: Optimizing the nucleotide sequence encoding recombinant protein C.

[0014] In order to increase the expression of recombinant protein C, on the premise that the amino acid sequence of recombinant protein C remains unchanged, the nucleotide sequence encoding recombinant protein C is optimized by using the most codon of Escherichia coli. The optimized nucleotide sequence is as follows:

[0015] CTGAACCTGAACAAACGCCTGCTGCATGAAACCCAGGCCCATGTGGATGATGCCCATCATGCCCATCATGTGGCCGATGCCCATCATGCCCATCTGAACCTGAACAAACGCCTGCTGCATGAAACCCAGGCCCATGTGGATGATGCCCATCATGCCCATCATGTGGCCGATGCCCATCATGCCCATGGCGGCGGCGGCGATGCCCGCCATGCCACCGATGCCCATCATGCCGCCGATGCCCATCATGCCACCGATGCCCATGATGCCCGCCATGCCACCGATGCCCATCATGCCGCCGATGCCCATCATGCCACCGATGCCCAT

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Abstract

The invention relates to a preparation method of HRPII protein monoclonal antibody of plasmodium falciparum. The preparation method comprises the following steps of: adopting HRPII protein of plasmodium falciparum as target antigen and respectively analyzing and selecting two dominant antigen epitopes of A and B; respectively repeating the two dominant antigen epitopes of A and B, then continuously connecting four glycine and forming recombinant protein C; adopting most securest code of escherichia coli and converting the amino acid sequence of the recombinant protein C into corresponding nucleotide sequence; carrying out chemical synthesis to the former step to obtain the nucleotide sequence, and respectively adding enzyme cutting sites BamHI and EcoRI at the upstream and downstream thereof; inserting nucleotide fragment obtained by the former step into expression carrier PET-28a(+), constructing recombinant protein C expression carrier and inducing to express the recombinant proteinC in the escherichia coli BL21 (DE3); carrying out ultrasonic bacteria breaking and low-temperature centrifugation, then taking supernatant of the solution, affining a chromatographic column by nickel-agarose, eluting and obtaining purified recombinant protein C; after immunizing Balb / c mouse with the recombinant protein C for a plurality of times, taking and fusing spleen cells with sp2 / 0 myelomacells, and obtaining six hybridoma cell lines by multiple rounds of screening; and purifying monoclonal antibody, respectively marking horse radish peroxidase and prorating matching and combination of optimum monoclonal antibody by ELISA orthogonal experiment.

Description

technical field [0001] The present invention relates to a method for tandem expression of dominant antigenic epitope of Plasmodium falciparum HRPII protein, preparation of monoclonal antibody to dominant antigenic epitope of Plasmodium falciparum HRPII protein and method for detection of Plasmodium falciparum by enzyme-linked immunosorbent assay The invention relates to a preparation method of HRPII protein monoclonal antibody, which belongs to the field of bioengineering technology manufacturing. Background technique [0002] At present, due to the influence of various factors, no effective malaria vaccine has been put into practical use so far, so it is particularly important to realize rapid diagnosis and early treatment of malaria. Although the traditional thick and thin blood film smear microscopy method is specific, it is time-consuming and laborious, and requires skilled operators, so this method is not suitable for the rapid diagnosis of malaria. The sensitivity of ...

Claims

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Application Information

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IPC IPC(8): C12P21/08C12N15/70C12P21/02C12N15/06C12R1/19
CPCY02A50/30
Inventor 余铭恩冯俊涛李晓照吴琼杉敖翔余卫
Owner 杭州新脉生物科技有限公司
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