CDNA cloning and expression of Rhodnius prolixus vitellogenin

Abstract

It was shown that Rhodnius prolixus vitellogenin (Vg) is synthesized as precursors of 205 and 190 kDa. Each Vg subunit is antigenically related to a domain in the precursor molecules. Since Vg has been previously detected in R. prolixus male adults, protein synthesis by fat instar male nymphs was investigated and no Vg synthesis could be detected. Also, a 6.1 Kb RNA is present in female adults but not in 5th instar male nymphs. Therefore, cDNAs from female adult and 5th instar male fat bodies were used for differential screening of a female fat body cDNA library leading to the isolation of several female specific clones. All the clones hybridizing to the female specific 6.1 Kb RNA species were identical. We also describe the construction of new expression vectors, pGex-A and pGex-B, derived from the previously described plasmid pGex-1N. The new vectors, together with pGex-3X, comprise a set of expression plasmids with cloning sites in all thee possible reading frames that give a fusion polypeptide with the glutathione S-transferase. This carrier protein can be cleaved by digestion with factor Xa in all three plasmids; one of the Vg cDNA clones was subcloned in pGex-A. Antibodies affinity purified from the fusion protein Vg/glutathione S-transferase recognized both large Vg subunits, suggesting an antigenic relationship between them. Furthermore, the small Vg subunits were not recognized, indicating that they may be localized at the N-terminal region of Vg precursors.