Cathepsin D is an aspartic protease mainly found in lysosomes. As a proteolytic enzyme, cathepsin B has been reported to play an essential role in the embryonic degradation of Vn in insects. Cathepsin B is a cysteine protease involved in a wide range of biological processes, including the degradation of regulatory proteins and digestive processes. Among them, three enzymes including cathepsin B, cathepsin D and acid phosphatase have been extensively investigated in insects. Regulatory mechanisms for the utilization of Vn have been implicated to rely on a variety of proteolytic enzymes in different species of insects. In ticks, vitellin (Vn) is a phosphorylated heme-lipoglycoprotein and its degraded products provide the source of amino acids, carbohydrates, heme, and other nutrients for embryonic development and unfed larva. Embryonic development in eggs, the only non-parasitic stage, is vital in the tick life-cycle. Therefore, one of the strategies to control tick-borne diseases is to explore new molecular targets for the interruption of the tick life-cycle. After engorgement, the body weight of female ticks is increased nearly 100-fold compared to unfed ticks, and more than 50% of engorgement weight is transformed into eggs. With a high reproductive potential, the fully engorged female can lay up to several thousand eggs. With climate change, new tick-borne pathogens and tick-borne diseases have become severe threats to public health all over the world. They are notorious vectors that can transmit various pathogens (viruses, rickettsiae, bacteria, spirochaetes and protozoans) among arthropods. Ticks are obligatory hematophagous ectoparasites of wildlife, domestic animals and humans. The profiles of both mRNA expression and enzymatic activity of these enzymes indicate that they are controlled orderly and play multiple roles during embryonic development in ticks. Three enzymes were expressed and activated in eggs, and also presented different dynamic changes with the development of embryos. Acid phosphatase activity increased gradually during the first five days and then remained stable until the end of egg development. The highest activity of cathepsin B was observed on the first day of egg development, whereas cathepsin D reached its highest activity on day 13. Cathepsin B reached its highest expression on day 5, whereas the peak expression of acid phosphatase and cathepsin D occurred on day 11. Both cathepsin B and acid phosphatase transcripts were accumulated during the first four days. The results revealed that all three enzymes were expressed throughout embryonic development. In the present study, the mRNA expression profiles and enzymatic activity of cathepsin B, cathepsin D and acid phosphatase were investigated during embryonic development in the tick Haemaphysalis longicornis. Table 2 is a complete list of the Flexi® Vectors and which sequencing primers have been used to sequence inserts cloned into each vector.Three main enzymes including cathepsin B, cathepsin D and acid phosphatase are involved in vitellin degradation, which is a major biochemical event of the embryonic development and can provide nutrients and metabolites for tick embryos. Listed in Table 1 are the sequencing primers used by Promega scientists to confirm insert sequences in the Flexi® Vectors. Taq DNA polymerase is not recommended for cloning protein-coding regions due to the relatively high error rate, but if this polymerase is used, 5–6 clones will typically need to be sequenced to find a 2 kilobase insert that is error-free. Even when using thermostable DNA polymerases with proofreading activity, such as Pfu DNA polymerase, it is still necessary to sequence 1–2 clones to ensure no errors were introduced during the PCR. These restriction enzyme sites are then used to clone the PCR product into the Flexi® Vector. When first creating a Flexi® Vector construct, the protein-coding region is amplified using primers that append SgfI and PmeI restriction sites on either end of the sequence. It is based on two rare-cutting restriction enzymes, SgfI and PmeI, and provides a rapid, efficient and high-fidelity way to transfer protein-coding regions between a variety of Flexi® Vectors without the need to resequence. The Flexi® Vector System is a simple, yet powerful, directional cloning method for protein-coding sequences (1).
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