| Haemonchus contortus is a highly pathogenic bloodsucking nematode,which lives in ruminant abomasum.A large number of parasites will consume the nutrition of the host for a long time,which will increase the cost of breeding and even cause the death of animals.rapid and accurate diagnosis is very important for the prevention and control of the disease.However,due to the limitations of long detection cycle,low sensitivity and the need for professional technicians,traditional diagnosis methods are difficult to make accurate diagnosis,especially in the early and mild infection of animals.Therefore,it has potential application value to study fast,sensitive and specific diagnosis methods.In recent years,it has been found that Haemonchus contortus excretes and secretes some proteins from the host body at different developmental stages,which are called excretory secretory proteins(ESPs).In our laboratory,Dr.javaid incubated with the peripheral blood mononuclear cells(PBMCs)of goats by using ESPs and LC-MS/MS,and got 140 kinds of binding proteins.Three of them were cloned and expressed,including Methyltransferase type 12 protein(Mt12),Elongation Factor 1α protein(EF-1α)and Ubiquinone Oxidoreductase Domain Protein(NDUDC);The reactivity of the recombinant protein with sera from goats infected with Haemonchus contortus at different stages was analyzed;On this basis,the indirect ELISA method was established and applied.It was found that Mt12,EF-1α and NDUDC recombinant proteins of Haemonchus contortus could bind to the serum of goat infected with Haemonchus contortus for 7 days,which was of early diagnostic value.The indirect ELISA method based on Mt12,EF-la and NDUDC has good sensitivity and specificity.It can be used to detect clinical samples,and the coincidence rate with the results of autopsy is more than 88%.1 Cloning,expression and bioinformatics analysis of gene MT12,EF-la and NDUDC from Haemonchus contortusBased on the gene sequence of Mt12(GenBank accession number CDJ87424.1),EF-1α(GenBank accession number CDJ84328.1)and NDUDC(GenBank accession number CDJ88764.1),a pair of specific primers were designed respectivly.The ORF of the corresponding gene was obtained by RT-PCR using the total RNA of Haemonchus contortus as the template.The PCR amplification products were recovered and purified,linked to the pMD-19t clone vector,selected a single colony for PCR and double enzyme digestion identification,and selected positive plasmids for sequencing analysis.The results showed that the ORF of the three genes were 930 bp,1395 bp and 438 bp,respectively,which were consistent with the reference gene sequence.The correct gene fragments were sequenced and subcloned into pET-3 2a expression vector.The prokaryotic expression plasmids pET-32a/Mtl2、pET-32a/EF-1α and pET-32a/NDUDC were constructed.Western blot analysis showed that Mtl2,EF-la and NDUDC,three recombinant proteins,could be specifically recognized by goat sera artificially infected with Haemonchus contortus,which indicated that these proteins could stimulate the host to produce corresponding antibodies and had good immunogenicity.The Mtl2 gene encodes 309 amino acids,with a theoretical isoelectric point of 6.75 and a relative molecular weight of 35.927kDa.Mtl2 is a hydrophilic protein(gravy=-0.547),which is 100%similar as Mtl2 of Haemonchus contortus in genbank,and 72%-94%similar as similar protein sequences of Brazilian Yen Nematode,Polycentric Spiral Nematode and Viviparous Net Tailed Nematode.The EF-1α gene encodes 464 amino acids,has a theoretical isoelectric point of 8.95 and a relative molecular weight of 50.566kDa.It is predicted that the hydrophobicity of EF-1α is a hydrophilic protein(gray=-0.307).The sequence of this protein is 99%similar as EF-1α of Haemonchus contortus in genbank,and 93%～98%similar to that of the analogues,such as Leptospira ceyloides,P.Americanus and P.Viviparum.The NDUDC gene encodes 145 amino acids,the theoretical isoelectric point is 11.06,and the relative molecular weight is 17kDa.The NDUDC is a hydrophilic protein(gravy=-0.861),which is 100%similar as the NDUDC of Haemonchus contortus in genbank,and 71%～86%similar as the protein sequences of Cryptostrongylus Elegans,Lepidoptera Ceyloides and Rhabdomus Animus.2 Analysis of early diagnostic potential of MT12,EF-1 and NDUDCGoats were artificially infected with the third stage larvae of Haemonchus contortus.The blood of goats at different stages(0,7,14,28,35,49,61,80 and 103 days)was collected to prepare serum.The serum of goats at different stages of infection was used as an antibody to analyze the three recombinant proteins of Mt12,EF-1α and NDUDC by Western blot.The results showed that the sera of 7～103 days after infection could react with EF-la and NDUDC recombinant protein;The recognition period of Mt12 protein was 7～61 days after goat infection.It is suggested that Mtl2,EF-la and NDUDC proteins have potential value in early diagnosis.3 Establishment of indirect ELISA based on Mt12,EF-1α and NDUDC proteinIn this study,three recombinant proteins,Mt12,EF-1α and NDUDC,were used as coating antigens to establish indirect ELISA.The conditions of antigen coating,serum reaction,blocking,second antibody action and critical value were explored to determine the best reaction conditions of indirect ELISA.The results showed that the best reaction conditions for indirect ELISA of Mt12 protein were as follows:1.25μg/mL of coating concentration,1:25 of serum dilution;60 min of the first antibody;2 h of sealing with 5%BSA at 37℃;60 mins of reaction with 1:8000 dilution at 37℃;0.40 of the positive and 0.36 of the second antibody,between the two(0.36<OD450<0.40)was false negative/positive.The best reaction conditions of EF-1α protein were:1.25 μg/mL coating concentration,1:25 serum dilution;the best reaction time of the first antibody was 60min;5%bovine serum albumin was sealed at 37℃ for 2h;the second antibody was diluted at 1:8000 for 60min at 37℃;the positive critical value of the test sample was 0.40,the negative critical value was 0.35,and between the two(0.35<OD450<0.40)it was false negative/positive.The optimal reaction conditions of NDUDC protein were as follows:coating concentration 0.625 μg/mL,serum dilution 1:25;the best reaction time of the first antibody was 90 mins;5%BSA was used to seal at 37 ℃ for 2h;the second antibody was used to react at 37℃ with 1:8000 dilution for 90 min to detect the OD450≥0.36,it is determined to be positive;otherwise,it is determined to be negative positive with a critical value of 0.36 and negative with a critical value of 0.32.Between the two(0.32<OD450<0.36),it is false negative/positive.The specific test showed that the three purified recombinant proteins could be used as coating antigens to detect the sera of goat infected with Haemonchus contortus,and the detection methods established by the repeatability test showed good repeatability.4 Comparison of indirect ELISA and other diagnostic methodsIn order to evaluate the feasibility of the established ELISA method in clinical detection,51 goats were randomly selected from different sheep farms in Nanjing,Jiangsu Province,and their serum,feces and abomasum samples were collected.Egg count,abomasum adult count and indirect ELISA were used to detect these samples,and the results of three diagnostic methods were compared.The abomasum section showed 10 positive samples and 41 negative samples.Ten positive samples,40 negative samples and one false negative/positive samples were detected by ELISA based on Mt12 protein,eight positive samples,37 negative/positive samples and six false negative/positive samples were detected by ELISA based on EF-1α protein;There were 7 positive samples,39 negative samples and 5 false negative/positive samples detected by ELISA based on NDUDC protein.Fecal examination showed that 8 samples were positive and 43 samples were negative.The results of indirect ELISA based on Mt12,EF-1α and NDUDC were 98%,88%and 90%,respectively. |
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