| Hc38 gene is abundantly expressed in intestinal microvilli of Haemonchus contortus . It is speculated that its products is a very important function protein, which may have a relation with blood feeding and escaping immune mechanism, however, the research of its function haven't been reported yet. The conserved domain of 196-512 amino acid belongs to the function-unknown Protein Family pf04910. 71 protein sequences of this family were logged in GeneBank, they distribute in 45 species, and 14 of these species are severe blood plague sources or mediums of human being or animals. In this research, by RNAi technique, the cDNA segment of conserved domain of Hc38 expression products was cloned, the RNAi vectors were constructed; and biology function of Hc38 gene was studied. This will be a foundation for looking for the blocking troche of Hc38 gene production domain and further reveal the function of Hc38 protein famlly that contained 196-512 gene amino acidses.The female adult of Haemonchus contortus were collected from abattoir in Shihezi. Then, the cDNA segments of 196-512 conservative domain fragment of Hc38 were cloned. After BLAST analysis, it 99% coincided with GenBank accession No. AY749124. This sequence had been submitted to GenBank under accession No. DQ414466.In following research, Hc38 segment was linked with L4440 vector to construct the RNAi vectors. The construction was successfully verified by PCR and cutting result of enzymes. Then, the RNAi vector was transferred into E.coli HT115, after induced by IPTG, the dsRNAs were introduced into the L3 larvae of Haemonchus contortus by feeding. 24 hours later, the RNAi group sheep and the control group were challenged by 70'000 Haemonchus contortus L3 respectively. EPG (eggs per gram feces) was tested on 21st, 23rd, 25th, 27th, 29th, 31st,33rd, 35th day respectively. At the same time, L3 stage larvae were hatched from eggs of 27th, 29th, 31st day. On the 35th day, the sheep was killed, the number and abnormality rate of imagoes from the stomachs of sheep were calculated. Worm burdens of lambs and worm length were tested.The result showed that comparing with the control group, EPG percentage of L3 hatched from eggs, and the number of female worms, male worms and adult worms reduced by 93.39%,86.70%,97.32%,98.58%,98.00% respectively, and the length of female and male decreased by 41.94%,19.86% respectively. In RNAi group, the percentage of abnormality of female worm is 15.2% and 83.3 % for male worm, however, in control group, there was only 2% in female and none for male.In conclusion: Once Hc38 was suppressed, it may cause blood feeding reducing, malnutrition, under-development, laying reduction and hatchability decrease in Haemonchus contortus. This was consistent with the hypothesis brought forward by Hartman, Australian. However, it may also be related to the function of corresponding muscle system or issue. Further research is necessary to clarify the physiological function of Hc38 gene. |
