Application of FoF1-ATPasebeta protein in promotion or reduction of adsorption of cells on apoptotic bodies
1. The technology of fof1-atpase and apoptotic body, which is applied in the fields of application, peptide/protein components, medical preparations containing active ingredients, etc., can solve the problems of failing to control the accumulation of apoptotic bodies
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Embodiment 1
[0062] 1.F o f 1 - Primer design for ATPaseβ
[0063] Transcriptome data from Spodoptera litura After Parasitization by Microplitis bicoloratus.PLoS ONE 9( 10): Select F from e110967.doi:10.1371 / journal.pone.0110967) o f 1-ATPaseβ gene sequence, F o f 1 - ATPaseβ gene sequence (shown as SEQ ID No.5) has a size of 1551 bp and encodes a total of 516 amino acids (SEQ ID No.6). Specific primers were designed using the primer design software Primer Premier 5. upstream primer F o f 1 -ATPase β-F (SEQ ID No.7) and downstream primer F o f 1 - ATPase β-R (SEQ ID No. 8).
[0064] 2. Extraction of total RNA and cDNA synthesis of Spodoptera litura larvae
[0065] In this experiment, the laboratory protocol was used to extract total RNA from Spodoptera litura larvae. The specific steps are as follows:
[0066] 1) Take 10 3-day-old larvae, place them in EP tubes with a small amount of RNAisoTM Plus lysate, mash and grind them thoroughly, continue to add RNAisoTM Plus lysate unt...
Embodiment 2
[0147] Saccharomyces cerevisiae F o f 1 -The genebank accession number of ATPaseβ is AAA34444.1, Caenorhabditis elegans (Caenorhabditis elegans) F o f 1 -The genebank accession number of ATPaseβ is P46561.2, Drosophila melanogaster F o f 1 -The genebank accession number of ATPaseβ is CAA50332.1, Spodoptera litura F o f 1 -The genebank accession number of ATPaseβ is AFY62978.1, Xenopus laevis F o f 1 - The genebank accession number of ATPaseβ is NP_001080126.1, Homo sapiens F o f 1 - The genebank accession number of ATPaseβ is NP_001677.2. For various representative species from low to high F o f 1 The amino acid sequence of -AT Paseβ carries out the sequence comparison of the Walk A module and the Walk B module, see Figure 4 . Depend on Figure 4 It can be seen that F o f 1 -WalkerA and WalkerB of ATPaseβ are highly conserved in various species.
Embodiment 3
[0149] Only replace pIZT-ATPaseβ with pIZT-ATPaseβ(K202-A), the difference between pIZT-ATPaseβ(K202-A) and pIZT-ATPaseβ lies in the expression of F o f 1 -The amino acid at position 202 of ATPaseβ was mutated from the original lysine (K) to alanine (A), that is, the conserved amino acid lysine (K) in the Walker A module was mutated into alanine (A).
[0150] The operation methods of extraction of total protein, detection of Western bolt, and significant difference analysis are the same as those in Example 1. In this example, glyceraldehyde-3-phosphate dehydrogenase was used as an internal reference.
[0151] see results Figure 5 . Depend on Figure 5 It can be seen that cells overexpressing pIZT-ATPaseβ(K202-A) after mutation have significantly reduced absorption of apoptotic bodies compared with cells overexpressing pIZT-ATPaseβ, indicating that F o f 1 -At least the lysine (K) of Walker A in ATPaseβ plays an important role in the process of apoptotic body uptake.
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