| Cystatin is a cysteine protease inhibitor that inhibits papain-like cysteine protease activity.There are four subfamilies,of which family 2 has antigenic insects and inhi bits apoptosis.During the growth and development of ticks,salivary glands,ovaries a nd other organs can secrete several families 2,and participate in physiological proces ses such as blood sucking,blood meal digestion,and embryo development.This stud y examined in detail the genes,expression and function of Hfcyst-1,Hfcyst-2,Hfcyst-3 and Hfcyst-4 in Haemaphysalis flava.1.The 5' and 3' ends of Contig3911,Contig8700,Contig7143 and Contig8976,w ere amplified by RACE method to obtain full-length c DNAs of Hfcyst-1,Hfcyst-2,H fcyst-3 and Hfcyst-4.Hfcyst-1 is 738 bp in length,open reading frame(ORF)is 429 bp,encoding protein 142aa;Hfcyst-2 is 715 bp in length,ORF is 426 bp,encoding protein 141aa;Hfcyst-3 is 722 bp in length,ORF is 426 bp,encoding protein 141aa;Hfcyst-4 has a full length of 776 bp and an ORF size of 426 bp,encoding a protei n of 141 aa.Structural analysis indicated that they belong to the Cystatin family2.2.The expression levels of Hfcyst-1,Hfcyst-2,Hfcyst-3 and Hfcyst-4 were determi ned by Real-time qantitative PCR.The results showed that the expression levels of l arve,numph and adult ticks increased in different stages of development;In addition to Hfcyst-2,the expression level of 1/4 engorged female adult ticks was highest;in different tissues,the expression of fully engorged female adult ticks salivary glands a nd ovaries was higher than that of semi-saturation.3.Hfcyst-3 was inserted into pET32 a,and the positive plasmid was transformed into BL21(DE3).The recombinant protein(rHfcyst-3)was successfully expressed by IPTG induction.SDS-PAGE electrophoresis showed that rHfcyst-3 was expressed as an insoluble inclusion body,and the relative mass of the product was 33 kDa.The assay showed that the renatured and purified recombinant protein had an inhibitory effect on Cathepsin S. |
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